Novel non-selective cation channel in neuronal cells and methods for treating brain swelling

ABSTRACT

The present invention is directed to therapeutic compounds, treatment methods, and kits affecting the NC Ca-ATP  channel of neural tissue, including neurons, glia and blood vessels within the nervous system, and methods of using same. The NC Ca-ATP  channel is newly expressed in neural tissue following injury such as ischemia, and is regulated by the sulfonylurea receptor SUR1, being inhibited by sulfonylurea compounds, e.g., glibenclamide and tolbutamide, and opened by diazoxide. Antagonists of the NC Ca-ATP  channel, including SUR1 antagonists, are useful in the prevention, diminution, and treatment of injured or diseased neural tissue, including astrocytes, neurons and capillary endothelial cells, that is due to ischemia, tissue trauma, brain swelling and increased tissue pressure, or other forms of brain or spinal cord disease or injury. Agonists of the NC Ca-ATP  channel may be are useful in the treatment neural tissue where damage or destruction of the tissue, such as a gliotic capsule, is desired.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Non-Provisional patentapplication Ser. No. 11/359,946 filed on Feb. 22, 2006, which is acontinuation-in-part of U.S. Non-Provisional patent application Ser. No.11/229,236 filed on Sep. 16, 2005, issued as U.S. Pat. No. 7,872,048 onJan. 18, 2011, which claims priority to U.S. Provisional PatentApplication No. 60/698,272 filed on Jul. 11, 2005 and to U.S.Provisional Application No. 60/610,758 filed Sep. 18, 2004, and U.S.Non-Provisional patent application Ser. No. 11/359,946 is acontinuation-in part of U.S. Non-Provisional application Ser. No.10/391,561, now abandoned, filed on Mar. 20, 2003, which claims priorityto U.S. Provisional Patent Application Ser. 60/365,933 filed on Mar. 20,2002, all of which are hereby incorporated by reference herein in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbersHL082517 and NS048260 awarded by The National Institutes of Health and aMerit Review grant from the United States Department of VeteransAffairs. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally regards the fields of cell biology,neurophysiology, and medicine. In particular, the present inventionrelates to a novel non-selective monovalent cationic ATP sensitive ionchannel (hereinafter referred to as the NC_(Ca-ATP) channel) that iscoupled to sulfonylurea receptor type 1 in neural cells, includingastrocytes, neurons and neural endothelial cells, to compounds andtreatments that may modulate NC_(Ca-ATP) channel activity, and to kitsincluding compounds useful for treatment of disease or injury conditionssuch as stroke or brain trauma.

BACKGROUND OF THE INVENTION

Injury to the nervous system has serious consequences. Followingtraumatic brain injury and stroke, the normal response of thesurrounding brain is to mount a cellular response that includesformation of reactive astrocytes that are believed to be important to“contain” and “clean-up” the injury site. Swelling of neural cells ispart of the cytotoxic or cell swelling response that characterizes braindamage in cerebral ischemia and traumatic brain injury, and is a majorcause of morbidity and mortality. See, Staub et al., 1993; Kimelberg etal., 1995. A number of mediators have been identified that initiateswelling of neural cells, including elevation of extracellular K⁺,acidosis, release of neurotransmitters and free fatty acids. See,Kempski et al., 1991; Rutledge and Kimelberg, 1996; Mongin et al., 1999.Cytotoxic edema is a well-recognized phenomenon clinically that causesbrain swelling, which worsens outcome and increases morbidity andmortality in brain injury and stroke.

Mechanisms underlying apoptotic death of reactive astrocytes and othercells have been studied. See, Tanaka et al., 2000; Yu et al., 2001. Themechanisms responsible for necrotic cell death of astrocytes, neuronsand neural endothelial cells have not been characterized. Apoptotic celldeath is preceded by cell shrinkage and net loss of K⁺. See, Yu et al.,1997; Yu et al., 1999. By contrast, in necrotic cell death, the plasmamembrane is ruptured, causing cytosolic contents to be released andthereby triggering tissue inflammation. See, Leist and Nicotera, 1997.Necrotic cell death may be more deleterious to nearby viable tissues,given the secondary inflammatory damage that is initiated.

Necrotic cell death is initiated by osmotic swelling following influx ofNa⁺, the major extracellular osmolyte. In most cell types, accumulationof Na⁺ intracellularly is regarded as a passive process that does notrequire activation of specific effectors but that is due instead todefective outward Na′ pumping under conditions of low intracellularadenosine triphosphate concentration ([ATP]i). See, Leist and Nicotera,1997; Trump et al., 1997. Cell blebbing or swelling, an indication ofintracellular Na′ overload, is generally regarded as an early sign ofnecrotic cell death. See, Leist and Nicotera, 1997; Majno and Joris,1995.

Inhibition of ATP synthesis or ATP depletion also causes neural cellswelling, blebbing and, if sufficiently severe, plasma membranedisruption and cell death. See, Jurkowitz-Alexander et al., 1993. Themechanisms of neural cell swelling associated with ATP-depletionremained incompletely characterized. See, Lomneth and Gruenstein, 1989;Juurlink et al., 1992; Rose et al., 1998.

One potential mechanism would be changes in Na⁺ and K⁺ concentration dueto inhibition of the Na⁺/K⁺-ATPase pump. However, an equivalent degreeof osmotic swelling induced by ouabain-mediated inhibition of theNa⁺/K⁺-ATPase pump in neural cells does not produce largedepolarization, blebbing or cell death. See, Jurkowitz-Alexander et al.,1992; Brismar and Collins, 1993. Failure of the Na⁺/K⁺-ATPase pump,therefore, is not the mechanism critical to swelling of neural cells.None of these studies have identified the cellular mechanisminstrumental in the cell swelling that is associated with brain damagein cerebral ischemia and traumatic brain injury and spinal cord injury.

One subtype of ATP sensitive cation channel is a non-selective cationchannel, that is sensitive to Ca²⁺ and ATP. More specifically, somenon-selective cation channels are activated by intracellular Ca²⁺([Ca²⁺]_(i)) and inhibited by intracellular ATP ([ATP]_(i)). AlthoughCa²⁺- and ATP-sensitive cation channels had been identified in a numberof non-neural cell types, they have not been identified in astrocytes orany other neural cells. See, Sturgess et al., 1987; Gray and Argent,1990; Rae et al., 1990; Champigny et al., 1991; Popp and Gogelein, 1992;Ono et al., 1994, each of which is hereby incorporated by reference inits entirety. These non-astrocyte channels comprise a heterogeneousgroup with incompletely defined characteristics. They exhibitsingle-channel conductances in the range of 25-35 pS, discriminatepoorly between Na⁺ and K⁺, are impermeable to anions, for the most partimpermeable to divalent cations, and they are blocked by similarconcentrations of the adenine nucleotides ATP, ADP and AMP on thecytoplasmic side. The function of these non-selective ATP sensitivecation channels in these non-neural cell types remains enigmatic, inpart because unphysiological concentrations of Ca²⁺ are generallyrequired for channel activation.

Another subtype of ATP sensitive cation channel is the ATP-sensitivepotassium channel (K_(ATP) channels) in pancreatic β cells. One class ofinsulin secretagogues, the antidiabetic sulfonylureas, is used toinhibit these K_(ATP) channels and stimulate insulin release in diabetesmellitus. See, Lebovitz, 1985. Antidiabetic sulfonylureas mediate theireffect on K_(ATP) channels via a high affinity sulfonylurea receptor(SUR). See, Panten et. al., 1989; Aguilar-Bryan et. al., 1995. Severalisoforms of the SUR, termed SUR1, SUR2A, SUR2B, and SUR2C, have beenidentified and cloned. See, Aguilar-Bryan et. al., 1995; Inagaki et.al., 1996; Isomoto et. al., 1996; Lawson, 2000. These receptors belongto the ATP-binding cassette (ABC) transporter family, of which thecystic fibrosis transmembrane conductance regulator (CFTR), another ionchannel modulator, is also a member. See, Higgins, 1992; Aguilar-Bryanet. al., 1995. Notably, the CFTR has major therapeutic importance, sinceits genetic absence causes cystic fibrosis, a fatal disease.

The sulfonylurea receptor imparts sensitivity to antidiabeticsulfonylureas such as glibenclamide and tolbutamide. Also, SUR isresponsible for activation of the potassium channel by a chemicallydiverse group of agents termed K⁺ channel openers (SUR-activators), suchas diazoxide, pinacidil, and cromakalin. See, Aguilar-Bryan et. al.,1995; Inagaki et. al., 1996; Isomoto et. al., 1996; Nichols et. al.,1996; Shyng et. al., 1997b. In various tissues, molecularly distinctSURs are coupled to distinct channel moieties to form different K_(ATP)channels with distinguishable physiological and pharmacologicalcharacteristics. The K_(ATP) channel in pancreatic β cells is formedfrom SUR1 linked with a K⁺ channel, whereas the cardiac and smoothmuscle K_(ATP) channels are formed from SUR2A and SUR2B, respectively,linked to K⁺ channels. See, Fujita and Kurachi, 2000.

Gliotic Capsule

The gliotic capsule that forms around a “foreign body” in the brain isan important, albeit neglected, biological system. On the one hand, thegliotic capsule represents the response of the brain to an injuriousstimulus—an attempt by the brain to wall off, isolate, dispose of, andotherwise protect itself from the foreign body. On the other hand, thegliotic capsule forms a potentially harmful mass of tissue from whichoriginates edema fluid that contributes to brain swelling, and whoseconstituent cells undergo cytotoxic edema, which adds further to brainswelling. Also, the gliotic capsule protects foreign cells fromimmunologic surveillance.

The essential elements involved in formation of a gliotic capsule appearto be uniform in many types of CNS pathology, be it a traumaticallyimplanted foreign body, a metastatic tumor, a brain abscess, orinfarcted necrotic tissue following a stroke. First, microglia andastrocytes become activated near the site of injury, with large,stellate-shaped GFAP-positive reactive astrocytes forming the mostprominent cellular component of the response. Secondly, the foreignnature of the entity is recognized, and the response is initiated tosurround and contain it. Although the concept of “foreign body”encompasses a large variety of pathological conditions, the responses inmost cases bear a great deal of similarity to one another.

The interface between the foreign body and the gliotic capsule, referredto as the inner zone of the gliotic capsule, appears to be of greatimportance in determining the overall response to injury.

Thus, a need exists for a physiological target instrumental in the cellswelling that is associated with brain damage in cerebral ischemia andtraumatic brain injury and in the consequent morbidity and mortality.There is also a need for specific treatments for the cytotoxic edemathat causes brain swelling, which worsens outcome and increasesmorbidity and mortality in brain injury and stroke. Other and furtherobjects, features, and advantages will be apparent from the followingdescription of the presently preferred embodiments of the invention,which are given for the purpose of disclosure.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery of a specificchannel, the NC_(Ca-ATP) channel, which, for example, is expressed inneurons, glia and neural endothelial cells after brain trauma. Thisunique non-selective cation channel is activated by intracellularcalcium and blocked by intracellular ATP (NC_(Ca-ATP) channel), and canbe expressed in neuronal cells, neuroglia cells (also termed glia, orglial cells, e.g., astrocyte, ependymal cell, oligodentrocyte andmicroglia) or neural endothelial cells (e.g., capillary endothelialcells) in which the cells have been or are exposed to a traumaticinsult, for example, an acute neuronal insult (e.g., hypoxia, ischemia,tissue compression, mechanical distortion, cerebral edema or cellswelling), toxic compounds or metabolites, an acute injury, cancer,brain abscess, etc.

More specifically, the NC_(Ca-ATP) channel of the present invention hasa single-channel conductance to potassium ion (K⁺) between 20 and 50 pS.The NC_(Ca-ATP) channel is also stimulated by Ca²⁺ on the cytoplasmicside of the cell membrane in a physiological concentration range, whereconcentration range is from 10⁻⁸ to 10⁻⁵ M. The NC_(Ca-ATP) channel isalso inhibited by cytoplasmic ATP in a physiological concentrationrange, where the concentration range is from 10⁻¹ to 10 M. TheNC_(Ca-ATP) channel is also permeable to the following cations; K⁺, Cs⁺,Li⁺, Na⁺; to the extent that the permeability ratio between any two ofthe cations is greater than 0.5 and less than 2.

More particularly, the present invention relates to the regulationand/or modulation of this NC_(Ca-ATP) channel and how its modulation canbe used to treat various diseases and/or conditions, for example acuteneuronal insults (e.g., stroke, an ischemic/hypoxic insult, a traumaticor mechanical injury) and diseases or conditions leading to formation ofa gliotic capsule. Yet further, the present invention relates to theregulation and/or modulation of this NC_(Ca-ATP) channel and its role inmaintaining or disrupting the integrity of the gliotic capsule. Themodulation and/or regulation of the channel results from administrationof an activator or agonist of the channel or an antagonist or inhibitorof the channel. Thus, depending upon the disease, a composition (anantagonist or inhibitor) is administered to block or inhibit the channelto prevent cell death, for example to treat cerebral edema that resultsfrom ischemia due to tissue trauma or to increased tissue pressure. Inthese instances the channel is blocked to prevent or reduce or modulatedepolarization of the cells. Alternatively, in order to treat or disrupta gliotic capsule, it is desirable to open or activate the channel byadministering an agonist or activator compound to cause celldepolarization resulting in cell death of diseased target cells.

In one aspect, the present invention provides novel methods of treatinga patient comprising administering a therapeutic compound that targets aunique non-selective cation channel activated by intracellular calciumand blocked by intracellular ATP (NC_(Ca-ATP) channel). In specificembodiments, the therapeutic compound may be an antagonist, and usesthereof in therapies, such as treatment of cerebral ischemia or edema,benefiting from blocking and/or inhibiting the NC_(Ca-ATP) channel. Infurther embodiments, where death of cells expressing the NC_(Ca-ATP)channel is desired for therapeutic purposes, the therapeutic compoundmay be an agonist. Compositions comprising agonists and/or antagonistsof the NC_(Ca-ATP) channel are also contemplated.

The invention also encompasses the use of such compounds andcompositions that modulate NC_(Ca-ATP) channel activity to treat brainswelling. For example, the present invention relates to methods for thetreatment of brain swelling that results from brain trauma or cerebralischemia, resulting in neural cell swelling, cell death, and an increasein transcapillary formation of ionic and vasogenic edema. Furtherprovided is a method of preventing brain swelling and the resultingbrain damage through the therapeutic use of antagonists to theNC_(Ca-ATP) channel. In one embodiment, the therapeutic antagonist canbe administered to or into the brain. Such administration to the brainincludes injection directly into the brain, particularly in the casewhere the brain has been rendered accessible to injection due to traumato the skull, for example. The invention further provides thetherapeutic use of sulfonylurea compounds as antagonists to theNC_(Ca-ATP) channel to prevent cell swelling in brain. In one embodimentthe sulfonylurea compound is glibenclamide. In another embodiment, thesulfonylurea compound is tolbutamide, or any of the other compounds thathave been found to promote insulin secretion by acting on KATP channelsin pancreatic β cells, as listed elsewhere herein.

The invention also encompasses agonists and antagonists of theNC_(Ca-ATP) channel, including small molecules, large molecules, andantibodies, as well as nucleotide sequences that can be used to inhibitNC_(Ca-ATP) channel gene expression (e.g., antisense and ribozymemolecules). An antagonist of the NC_(Ca-ATP) channel includes one ormore compounds capable of (1) blocking the channel; (2) preventingchannel opening; (3) reducing the magnitude of membrane current throughthe channel; (4) inhibiting transcriptional expression of the channel;and/or (5) inhibiting post-translational assembly and/or trafficking ofchannel subunits.

The invention relates to assays designed to screen for compounds orcompositions that modulate the NC_(Ca-ATP) channel, particularlycompounds or compositions that act as antagonists of the channel, andthereby modulate neural cell swelling and the concomitant brainswelling. To this end, cell-based assays or non-cell based assays can beused to detect compounds that interact with, e.g., bind to, the outside(i.e., extracellular domain) of the NC_(Ca-ATP) channel and/or itsassociated SUR1 regulatory subunit. The cell-based assays have theadvantage in that they can be used to identify compounds that affectNC_(Ca-ATP) channel biological activity (i.e., depolarization). Theinvention also provides a method of screening for and identifyingantagonists of the NC_(Ca-ATP) channel, by contacting neural cells witha test compound and determining whether the test compound inhibits theactivity of the NC_(Ca-ATP) channel. In one embodiment, methods foridentifying compounds that are antagonists of the NC_(Ca-ATP) areprovided. In one embodiment, therapeutic compounds of the presentinvention, including NC_(Ca-ATP) antagonists, are identified by thecompound's ability to block the open channel or to prevent channelopening, such as by quantifying channel function usingelectrophysiological techniques to measure membrane current through thechannel, for example. NC_(Ca-ATP) antagonists include compounds that areNC_(Ca-ATP) channel inhibitors, NC_(Ca-ATP) channel blockers, SUR1antagonists, SUR1 inhibitors, and/or compounds that reduce the magnitudeof membrane current through the channel, for example. In thisembodiment, channel function can be measured in a preparation of neuralcells from a human or animal, and the test compound can be brought intocontact with the cell preparation by washing it over the cellpreparation in solution. The invention further provides a method ofscreening for sulfonylurea compounds that may act as antagonists of theNC_(Ca-ATP) channel.

The present invention relates to drug screening assays to identifycompounds for the treatment of brain swelling, such as the swelling thatoccurs after brain injury or cerebral ischemia by using the NC_(Ca-ATP)channel as a target. The invention also relates to compounds thatmodulate neural cell swelling via the NC_(Ca-ATP) channel. The presentinvention also relates to the treatment of brain swelling by targetingthe NC_(Ca-ATP) channel.

The present invention is also directed to purified compositionscomprising a novel Ca²⁺-activated, [ATP]_(i-) sensitive nonspecificcation channel. In a preferred embodiment of the present invention, thecompositions comprise mammalian neural cells or membrane preparationsexpressing the NC_(Ca-ATP) channel, most preferably wherein themammalian neural cells are freshly isolated reactive astrocytes, neuronsor neural endothelial cells. A preferred example of such a purifiedcomposition comprising the NC_(Ca-ATP) channel is a membrane preparationderived from native reactive astrocytes. As demonstrated herein, whenneural cells expressing the NC_(Ca-ATP) channel are depleted ofintracellular ATP, the NC_(Ca-ATP) channel opens and the cells swell anddie. However, if the NC_(Ca-ATP) channel is blocked on such cells, thecells do not swell and die. The invention is also based, in part, on thediscovery that the NC_(Ca-ATP) channel is regulated by a type 1sulfonylurea receptor, and that antagonists of this receptor are capableof blocking the NC_(Ca-ATP) channel and inhibit neural cell swelling.

The composition(s) of the present invention may be deliveredalimentarily or parenterally. Examples of alimentary administrationinclude, but are not limited to orally, buccally, rectally, orsublingually. Parenteral administration can include, but are not limitedto intramuscularly, subcutaneously, intraperitoneally, intravenously,intratumorally, intraarterially, intraventricularly, intracavity,intravesical, intrathecal, or intrapleural. Other modes ofadministration may also include topically, mucosally, transdermally,direct injection into the brain parenchyma.

An effective amount of an agonist or antagonist of NC_(Ca-ATP) channelthat may be administered to a cell includes a dose of about 0.0001 nM toabout 2000 μM, for example. More specifically, doses of an agonist to beadministered are from about 0.01 nM to about 2000 μM; about 0.01 μM toabout 0.05 μM; about 0.05 μM to about 1.0 μM; about 1.0 μM to about 1.5μM; about 1.5 μM to about 2.0 μM; about 2.0 μM to about 3.0 μM; about3.0 μM to about 4.0 μM; about 4.0 μM to about 5.0 μM; about 5.0 μM toabout 10 μM; about 10 μM to about 50 μM; about 50 μM to about 100 μM;about 100 μM to about 200 μM; about 200 μM to about 300 μM; about 300 μMto about 500 μM; about 500 μM to about 1000 μM; about 1000 μM to about1500 μM and about 1500 μM to about 2000 μM, for example. Of course, allof these amounts are exemplary, and any amount in-between these pointsis also expected to be of use in the invention.

An effective amount of an agonist or antagonist of the NC_(Ca-ATP)channel or related-compounds thereof as a treatment varies dependingupon the host treated and the particular mode of administration. In oneembodiment of the invention the dose range of the agonist or antagonistof the NC_(Ca-ATP) channel or related-compounds thereof will be about0.01 μg/kg body weight to about 20,000 μg/kg body weight. The term “bodyweight” is applicable when an animal is being treated. When isolatedcells are being treated, “body weight” as used herein should read tomean “total cell body weight”. The term “total body weight” may be usedto apply to both isolated cell and animal treatment. All concentrationsand treatment levels are expressed as “body weight” or simply “kg” inthis application are also considered to cover the analogous “total cellbody weight” and “total body weight” concentrations. However, those ofskill will recognize the utility of a variety of dosage range, forexample, 0.01 μg/kg body weight to 20,000 μg/kg body weight, 0.02 μg/kgbody weight to 15,000 μg/kg body weight, 0.03 μg/kg body weight to10,000 μg/kg body weight, 0.04 μg/kg body weight to 5,000 μg/kg bodyweight, 0.05 μg/kg body weight to 2,500 μg/kg body weight, 0.06 μg/kgbody weight to 1,000 μg/kg body weight, 0.07 μg/kg body weight to 500μg/kg body weight, 0.08 μg/kg body weight to 400 μg/kg body weight, 0.09μg/kg body weight to 200 μg/kg body weight or 0.1 μg/kg body weight to100 μg/kg body weight. Further, those of skill will recognize that avariety of different dosage levels will be of use, for example, 0.0001μg/kg, 0.0002 μg/kg, 0.0003 μg/kg, 0.0004 μg/kg, 0.005 μg/kg, 0.0007μg/kg, 0.001 μg/kg, 0.1 μg/kg, 1.0 μg/kg, 1.5 μg/kg, 2.0 μg/kg, 5.0μg/kg, 10.0 μg/kg, 15.0 μg/kg, 30.0 μg/kg, 50 μg/kg, 75 μg/kg, 80 μg/kg,90 μg/kg, 100 μg/kg, 120 μg/kg, 140 μg/kg, 150 μg/kg, 160 μg/kg, 180μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg,350 μg/kg, 375 μg/kg, 400 μg/kg, 450 μg/kg, 500 μg/kg, 550 μg/kg, 600μg/kg, 700 μg/kg, 750 μg/kg, 800 μg/kg, 900 μg/kg, 1 mg/kg, 5 mg/kg, 10mg/kg, 12 mg/kg, 15 mg/kg, 20 mg/kg, and/or 30 mg/kg. In particularembodiments, there may be dosing of from very low ranges (e.g. 1mg/kg/day or less; 5 mg/kg bolus; or 1 mg/kg/day) to moderate doses(e.g. 2 mg bolus, 15 mg/day) to high doses (e.g. 5 mg bolus, 30-40mg/day; and even higher). Of course, all of these dosages are exemplary,and any dosage in-between these points is also expected to be of use inthe invention. Any of the above dosage ranges or dosage levels may beemployed for an agonist or antagonist, or both, of NC_(Ca-ATP) channelor related-compounds thereof.

The NC_(Ca-ATP) channel is blocked by antagonists of type 1 sulfonylureareceptor (SUR1) and is opened by SUR1 activators. More specifically, theantagonists of type 1 sulfonylurea receptor (SUR1) include blockers ofK_(ATP) channels and the SUR1 activators include activators of K_(ATP)channels. The channel can be inhibited by an NC_(Ca-ATP) channelinhibitor, an NC_(CaATP) channel blocker, a type 1 sulfonylurea receptor(SUR1) antagonist, SUR1 inhibitor, or a compound capable of reducing themagnitude of membrane current through the channel. More specifically,the SUR1 antagonist may be selected from the group consisting ofglibenclamide, tolbutamide, repaglinide, nateglinide, meglitinide,midaglizole, LY397364, LY389382, gliclazide (also known in the art asglyclazide), glimepiride, estrogen, estrogen related-compounds(estradiol, estrone, estriol, genistein, non-steroidal estrogen (e.g.,diethystilbestrol), phytoestrogen (e.g., coumestrol), zearalenone,etc.), and compounds known to inhibit or block K_(ATP) channels. MgADPcan also be used to inhibit the channel. Other compounds that can beused to block or inhibit K_(ATP) channels include, but are not limitedto tolbutamide, glyburide (1[p-2[5-chloro-O-anisamido)ethyl]phenyl]sulfonyl]-3-cyclohexyl-3-urea);chlorpropamide (1-[[(p-chlorophenyl)sulfonyl]-3-propylurea; glipizide(1-cyclohexyl-3[[p-[2(5-methylpyrazinecarboxamido)ethyl]phenyl]sulfonyl]urea); or tolazamide(benzenesulfonamide-N-[[(hexahydro-1H-azepin-1yl)amino]carbonyl]-4-methyl).In additional embodiments, non-sulfonyl urea compounds, such as2,3-butanedione and 5-hydroxydecanoic acid, quinine, and therapeuticallyequivalent salts and derivatives thereof, may be employed in theinvention.

The channel is expressed on neuronal cells, neuroglia cells, neuralepithelial cells, neural endothelial cells, or a combination thereof,for example. The inhibitor blocks the influx of Na⁺ into the cellsthereby preventing depolarization of the cells Inhibition of the influxof Na⁺ into the cells thereby at least prevents or reduces cytotoxicedema and/or ionic edema, and prevents or reduces hemorrhagicconversion. Thus, this treatment reduces cell death or necrotic death ofneuronal and/or neural endothelial cells.

In certain embodiments, the amount of the SUR1 antagonist administeredto the subject is in the range of about 0.0001 μg/kg/day to about 20mg/kg/day, about 0.01 μg/kg/day to about 100 μg/kg/day, or about 100μg/kg/day to about 20 mg/kg/day. Still further, the SUR1 antagonist maybe administered to the subject in the from of a treatment in which thetreatment may comprise the amount of the SUR1 antagonist or the dose ofthe SUR1 antagonist that is administered per day (1, 2, 3, 4, etc.),week (1, 2, 3, 4, 5, etc.), month (1, 2, 3, 4, 5, etc.), etc. Treatmentsmay be administered such that the amount of SUR1 antagonist administeredto the subject is in the range of about 0.0001 μg/kg/treatment to about20 mg/kg/treatment, about 0.01 μg/kg/treatment to about 100μg/kg/treatment, or about 100 μg/kg/treatment to about 20mg/kg/treatment.

Another embodiment of the present invention comprises a method ofreducing mortality of a subject suffering from a stroke comprisingadministering to the subject a compound effective to inhibit NC_(Ca-ATP)channels in a neuronal cell, a neuroglia cell, a neural endothelial cellor a combination thereof. The compound reduces stroke size and reducesedema located in the peri-infarct tissue. The compound can beadministered alimentary (e.g., orally, buccally, rectally orsublingually) or parenterally (e.g., intravenously, intradermally,intramuscularly, intraarterially, intrathecally, subcutaneously,intraperitoneally, intraventricularly) and/or topically (e.g.,transdermally), mucosally, or by direct injection into the brainparenchyma.

Still further, another embodiment comprises a method of reducing edemain a peri-infarct tissue area of a subject comprising administering tothe subject a compound effective to inhibit NC_(Ca-ATP) channels in aneuronal cell, a neuroglial cell, a neural endothelial cell, or acombination thereof.

Further embodiments comprises a method of treating a subject at risk fordeveloping a stroke comprising administering to the subject a compoundeffective to inhibit a NC_(Ca-ATP) channel in neuronal cell, a neurogliacell, a neural endothelial cell or a combination thereof.

In certain embodiments, the subject is undergoing treatment for acardiac condition, thus the condition increases the subjects risk fordeveloping a stroke. The treatment, for example, may comprise the use ofthrombolytic agents to treat myocardial infarctions. Still further, thesubject may be at risk for developing a stroke because the subjectsuffers from atrial fibrillation or a clotting disorder. Other subjectsthat are at risk for developing a stroke include subjects that are atrisk of developing pulmonary emboli, subjects undergoing surgery (e.g.,vascular surgery or neurological surgery), or subjects undergoingtreatments that increase their risk for developing a stroke, forexample, the treatment may comprise cerebral/endovascular treatment,angiography or stent placement. In other embodiments, the subject may beundergoing treatment for vascular disease that could place the spinalcord at risk for ischemia, such as surgery requiring aorticcross-clamping, surgery for abdominal aortic aneurysm, etc. In otherembodiments, the patient may be undergoing surgery for a spinal orspinal cord condition, including discectomy, fusion, laminectomy,extradural or intradural surgery for tumor or mass etc., that wouldplace the spinal cord at risk of injury. In some embodiments of theinvention, the subject has a chronic condition, whereas in otherembodiments of the invention, the subject does not have a chroniccondition, such as a short-term condition.

Another embodiment of the present invention comprises a method oftreating a subject at risk for developing cerebral edema comprisingadministering to the subject a compound effective to inhibit aNC_(Ca-ATP) channel in a neuronal cell, a neuroglia cell, a neuralendothelial cell or a combination thereof. The subject at risk may besuffering from an arterior-venous malformation, or a mass-occupyinglesion (e.g., hematoma) or may be involved in activities that have anincreased risk of brain trauma.

Another embodiment of the present invention comprises a compositioncomprising a membrane preparation derived from a neural endothelial cellexpressing a NC_(Ca-ATP) channel, wherein channel is blocked byantagonists of type 1 sulfonylurea receptor (SUR1) and opened by SUR1activators. More specifically, the channel has the followingcharacteristics: (a) it is a 35 pS type channel; (b) it is stimulated bycytoplasmic Ca²⁺ in the concentration range from about 10⁻⁸ to about10⁻⁵ M; (c) it opens when cytoplasmic ATP is less than about 0.8 μM; and(d) it is permeable to the monovalent cations K⁺, Cs⁺, Li⁺ and Na⁺.

In further embodiments, the compound that inhibits the NC_(Ca-ATP)channel can be administered in combination with a thrombolytic agent(e.g., tissue plasminogen activator (tPA), urokinase, prourokinase,streptokinase, anistreplase, reteplase, tenecteplase), an anticoagulantor antiplatelet (e.g., aspirin, warfarin or coumadin), statins,diuretics, vasodilators (e.g., nitroglycerin), mannitol, diazoxide orsimilar compounds that stimulate or promote ischemic precondition.

Yet further, another embodiment of the present invention comprises apharmaceutical composition comprising a thrombolytic agent (e.g., tissueplasminogen activator (tPA), urokinase, prourokinase, streptokinase,anistreplase, reteplase, tenecteplase), an anticoagulant or antiplatelet(e.g., aspirin, warfarin or coumadin), statins, diuretics, vasodilators,mannitol, diazoxide or similar compounds that stimulate or promoteischemic precondition or a pharmaceutically acceptable salt thereof anda compound that inhibits a NC_(Ca-ATP) channel or a pharmaceuticallyacceptable salt thereof. This pharmaceutical composition can beconsidered neuroprotective, in specific embodiments. For example, thepharmaceutical composition comprising a combination of the thrombolyticagent and a compound that inhibits a NC_(Ca-ATP) channel isneuroprotective because it increases the therapeutic window for theadministration of the thrombolytic agent by several hours; for examplethe therapeutic window for administration of thrombolytic agents may beincreased by several hours (e.g. about 4-about 8 hrs) byco-administering antagonist of the NC_(Ca-ATP) channel.

Still further, another embodiment comprises a method of treating acutecerebral ischemia in a subject comprising administering to a subject anamount of a thrombolytic agent or a pharmaceutically acceptable saltthereof in combination with an amount of a compound that inhibits aNC_(Ca-ATP) channel or a pharmaceutically acceptable salt thereof. Incertain embodiments, the thrombolytic agent is a tissue plasminogenactivator (tPA), urokinase, prourokinase, streptokinase, anistreplase,reteplase, tenecteplase or any combination thereof. The SUR1 antagonistcan be administered by any standard parenteral or alimentary route, forexample the SUR1 antagonist may be administered as a bolus injection oras an infusion or a combination thereof.

Another embodiment of the present invention comprises a method ofdisrupting a gliotic capsule, such as to disrupt the integrity of thetumor-brain barrier surrounding a tumor in the brain of a subjectcomprising administering to the subject a compound effective to activatea NC_(Ca-ATP) channel in a neuronal cell, or a neuroglia cell, a neuralendothelial cell or a combination thereof.

Where destruction of cells expressing the NC_(Ca-ATP) channel isdesired, an SUR1 activator or agonist may be administered, for example,to reduce or remove a gliotic capsule. The activator compound or agonistcan be a type 1 sulfonylurea receptor agonist. For example, agoniststhat can be used in the present invention include, but are not limitedto agonist of SUR1, for example, diazoxide, pinacidil, P1075,cromakalin, or combinations thereof. Other agonists can include, but arenot limited to diazoxide derivatives, for example3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide (NNC55-9216), 6,7-dichloro-3-isopropylamino-4H-1,2,4-benzothiadiazine1,1-dioxide (BPDZ 154),7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (BPDZ73), 6-Chloro-3-isopropylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine1,1-dioxide (NNC 55-0118)4,6-chloro-3-(1-methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine1,1-dioxide (NN414),3-(3-methyl-2-butylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide(BPDZ 44),3-(1′,2′,2′-trimethylpropyl)amino-4H-pyrido(4,3-e)-1,2,4-thiadiazine1,1-dioxide (BPDZ 62), 3-(1′,2′,2′-trimethylpropyl)amine-4H-pyrido(2,3-e)-1,2,4-thiadiazine, 1,1-dioxide (BPDZ 79),2-alkyl-3-alkylamino-2H-benzo- and2-alkyl-3-alkylamino-2H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides,6-Chloro-3-alkylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxidederivatives, 4-N-Substituted and -unsubstituted 3-alkyl- and3-(alkylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides, orcombinations thereof. In addition, other compounds, including6-chloro-2-methylquinolin-4(1H)-one (HEI 713) and LN 533021, as well asthe class of drugs, arylcyanoguanidines, are known activators or agonistof SUR1. Other compounds that can be used include compounds known toactivate K_(ATP) channels.

Still further, another embodiment of the present invention comprises amethod of inducing cell death of one or more of a neuronal or aneuroglia cell or a neural endothelial cell comprising administering tothe cell a compound effective to activate a NC_(Ca-ATP) channel in thecell. Activation of the NC_(Ca-ATP) channel results in an influx ofsodium ions (Na⁺) causing depolarization of the cell. The influx of Na⁺alters the osmotic gradient causing an influx of water into the cellthat leads to cytotoxic edema ultimately resulting in necrotic celldeath.

Yet further, another embodiment of the present invention comprises amethod of maintaining the integrity of the gliotic capsule surroundingbrain abscess of a subject comprising administering to the subject acompound effective to inhibit and/or block at least one NC_(Ca-ATP)channel in a neuronal cell, a neuroglia cell, a neural endothelial cellor a combination thereof.

Still further, another method of the present invention comprises amethod of diagnosing neuronal cell edema and/or cytotoxic damage in thebrain comprising: labeling an antagonist of SUR1; administering thelabeled antagonist of SUR1 to a subject; measuring the levels of labeledantagonist of SUR1 in the brain of the subject, wherein the presence oflabeled antagonist of SUR1 indicates neuronal cell edema and/orcytotoxic damage in the brain.

In further embodiments, the methods can comprise a method of determiningthe penumbra following a stroke comprising: labeling an antagonist ofSUR1; administering the labeled antagonist of SUR1 to a subject;visualizing the labeled antagonist of SUR1 in the brain of the subject,wherein the presence of labeled antagonist of SUR1 indicates thepenumbra.

Yet further, the present invention comprises a method monitoring strokeneural disease comprising: labeling an antagonist of SUR1; administeringthe labeled antagonist of SUR1 to a subject; visualizing the labeledantagonist of SUR1 in the brain of the subject, wherein the presence oflabeled antagonist of SUR1 indicates the progression of the disease. Incertain embodiments, the step of visualizing is performed daily tomonitor the progression of the stroke.

Another embodiment comprises a neuroprotective infusion kit comprising acompound that inhibits a NC_(Ca-ATP) channel in a neuronal cell, aneuroglia cell, a neural endothelial cell or a combination thereof andan IV solution. The compound and solution are contained within the samecontainer or within different containers. More specifically, thecompound is contained within the container of solution.

The kit may further comprise a neuroprotective bolus kit, wherein thebolus kit comprises a pre-loaded syringe of a compound inhibits aNC_(Ca-ATP) channel in a neuronal cell, a neuroglia cell, a neuralendothelial cell or a combination thereof.

Still further, another embodiment comprises a neuroprotective kitcomprising a compound that inhibits NC_(Ca-ATP) channel in a neuronalcell, a neuroglia cell, an endothelium cell or a combination thereof anda thrombolytic agent (e.g., tPA), an anticoagulant (e.g., warfarin orcoumadin), an antiplatelet (e.g., aspirin), a diuretic (e.g., mannitol),a statin, or a vasodilator (e.g., nitroglycerin).

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F. FIG. 1A shows whole cell current clamp recording before andafter exposure to ouabain and before and after exposure to NaN₃. FIG. 1Bshows whole cell voltage-clamp recordings during ramp pulses (a) beforeand (b) after exposure to NaN₃; (c) is the difference current. FIG. 1Cshows whole cell voltage-clamp recordings during step pulses (a) beforeand (b) after exposure to NaN₃; (c) is the difference current. FIG. 1Dshows cell-attached patch recording of single ion channel openingsinduced by NaN₃ at membrane potentials of (3) −80 mV and (4) 80 mV,compared to control patches at membrane potentials of (1) 80 mV and (2)−80 mV. FIG. 1E shows the cell-attached patch currents of FIG. 1D, shownat higher time resolution. FIG. 1F shows the cell-attached patchsingle-channel current-voltage relationship.

FIGS. 2A and 2B. FIG. 2A shows single channel currents recorded in aninside-out patch at different membrane potentials; dotted line indicateschannel closing. FIG. 2B is a plot of inside-out patch single channelamplitude vs. membrane potentials.

FIGS. 3A-3D. FIG. 3A shows single channel currents recorded in aninside-out patch with various alkaline ions substituting for K⁺ in thepipette; dotted line indicates channel closing. FIG. 3B is a plot ofchannel amplitude vs. membrane potential with various alkaline ionssubstituting for K⁺ in the pipette. FIG. 3C is a plot of channelamplitude measured in inside-out patches vs. voltage with Ca2⁺ and Mg²⁺substituting for K⁺ in the pipette. To estimate channel pore size, FIG.3D is a plot illustrating the relationship between the permeability(relative to Cs⁺) and the molecular radius of a series of monovalentorganic cations, which included: (a) methanolmine, (b) guanidium, (c)ethanolamine, (d) diethylamine, (e) piperazine, (f) Tris, and (g)N-methylglucamine, data indicating an equivalent pore size of 0.67 nm.

FIGS. 4A and 4B. FIG. 4A shows single channel recordings in aninside-out patch in the absence and presence of cytoplasmic ATP. FIG. 4Bis a plot of normalized open channel probability (n·Po) vs.concentration of cytoplasmic ATP.

FIGS. 5A and 5B. FIG. 5A shows current records from an inside-out patchexposed to different concentrations of [Ca²⁺]_(i). FIG. 5B the values ofn·Po measured at the membrane potentials and [Ca²⁺]_(i) indicated.

FIG. 6 is a plot of mean single channel amplitudes obtained in aninside-out patch configuration at different potentials studied and withdifferent [Mg²⁺]_(i); the dotted line indicates 35 pS conductance.

FIGS. 7A and 7B show that presence of SUR1 mRNA and absences of Kir6.1and Kir 6.2 in reactive astrocytes. Lanes 3 and 5 in FIG. 7A show thepresence of SUR1 in insulinoma RIN-m5f cells and NRAs, respectively.Lanes 4 and 6 in FIG. 7A show that SUR2 is absent in both cell types.Lanes 3 and 4 in FIG. 7B show that Kir6.1 is present in insulinomaRIN-m5f cells and Kir6.2 is absent from the insulinoma cells,respectively. Lanes 5 and 6 in FIG. 7B show that neither Kir6.1 norKir6.2 is present in NRAs, respectively.

FIG. 8 shows current recordings in an inside-out patch to illustrate theeffects of tryptic digestion on channel sensitivity to glibenclamide andATP.

FIGS. 9A and 9B shows that the channel activator diazoxide can elicitchannel activities under outside-out patch recording configuration. FIG.9A shows the outside-out patch recordings with Na azide and diazoxideapplied to the extracellular side of the membrane. FIG. 9B shows thecurrent records obtained from the segments marked with the correspondingnumbers in FIG. 9A, at higher temporal resolution.

FIGS. 10A-10C. FIG. 10A shows outside-out patch recordings (a) before,(b) during, and (c) after application of glibenclamide to theextracellular side of the membrane. FIG. 10B shows the current recordsof FIG. 10A at higher temporal resolution. FIG. 10C show a plot of meansingle channel amplitudes at the different potentials studied; the slopeof the data indicates 35 pS conductance of the glibenclamide-sensitivechannel.

FIGS. 11A and 11B show that sulfonylurea compounds inhibit channelactivities. FIG. 11A shows the outside-out patch recordings with variousconcentrations of tolbutamide applied to the extracellular side of themembrane. FIG. 11B shows the dose-response curves for inhibition of openchannel probability by glibenclamide and tolbutamide to provide anormalized open channel probability (n·Po); data were fit to a standardlogistic equation, with a Hill coefficient of 1 and half-maximuminhibition of 48 nM and 16.1 μM; values plotted are means (^(±)SE) from3 and 5 patches for Glibenclamide and Tolbutamide, respectively.

FIGS. 12A, 12B and 12C show the probability of channel opening in thepresence of 0 μM, 3 μM, and 30 μM tolbutamide, respectively.

FIGS. 12D, 12E and 12F show the distribution of open channel dwell timesin the presence of 0 μM, 3 μM, and 30 μM tolbutamide, respectively.

FIGS. 12G, 12H and 12I show the distribution of closed channel dwelltimes in the presence of 0 μM, 3 μM, and 30 μM tolbutamide,respectively.

FIG. 13A shows outside-out patch recordings with diazoxide applied tothe extracellular side of the membrane.

FIG. 13B shows current records at higher temporal resolution afterapplication of diazoxide and at different membrane potentials.

FIG. 13C shows a plot of mean single channel amplitudes at the differentpotentials studied; the slope indicates 35 pS conductance ofglibenclamide-sensitive channel.

FIGS. 14A, 14B and 14C are scanning electron micrographs of freshlyisolated native reactive astrocytes. FIG. 14A shows the cells whenformaldehyde-glutaraldehyde fixation was initiated under controlconditions; FIG. 14B shows the cells fixed 5 min after exposure to 1 mMNaN₃. FIG. 14C shows the cells fixed 25 min after exposure to 1 mM NaN₃.Bar, 12 μm.

FIGS. 15A, 15B and 15C. FIG. 15A has photomicrographs of theepifluorescence images of cells exposed to different compounds andlabeled with propidium iodide (upper panel a, b and c) or annexin V(lower panel d, e and f). The compounds were: control (a & d), 1 mM Naazide (b & e), 1 mM Na azide plus 1 μM glibenclamide (c & f). FIG. 15Bhas bar graphs showing cell-counts for propidium iodide labeling;pairwise multiple comparisons indicated a significant difference(p<0.05) with Na azide treatment; FIG. 15C has bar graphs showingcell-counts for annexin V staining; pairwise multiple comparisonsindicated no significant difference with any treatment.

FIG. 16 shows that addition of exogenousphosphatidylinositol-4,5-bisphosphate (PIP₂) causes activation of theNC_(Ca-ATP) channel, despite the presence of ATP in the bath solution.Initially, channel activity was recorded in an inside-out patch ofmembrane from an R1 astrocyte, with a bath solution containing 1 μM Ca²⁺and 10 μM ATP, which was sufficient to block channel activity. Additionof 50 μM PIP₂ resulted in channel activation, reflecting an apparentdecrease in affinity of the channel for ATP.

FIG. 17 shows that the NC_(Ca-ATP) channel in an R1 astrocyte isinhibited by estrogen. The initial portion of the record shows briskactivity from a number of superimposed channels, recorded in a cellattached patch of membrane from an R1 astrocyte obtained from a female.Addition of 10 nM estrogen to the bath promptly resulted in stronginhibition of channel activity. The mechanism involved is believed to berelated to estrogen receptor mediated activation of phospholipase C(PLC), resulting in depletion of PIP₂ from the membrane, and reflectingan apparent increase in affinity for ATP.

FIGS. 18A and 18B show Western blots demonstrating that R1 astrocytesfrom both males and females express estrogen receptors and SUR1, amarker of the NC_(Ca-ATP) channel. Cell lysates were obtained fromgelatin sponge implants from males (M) and females (F) and studied attwo dilutions (4× and 1×), with lysates from uterus used as controls.FIG. 18A was developed using antibodies directed against estrogenreceptors (ER), demonstrating that both ERα and ERβ are expressed inastrocytes from both genders. Western blots showed that SUR1 is alsoexpressed by cells from both genders, with pancreatic tissue used ascontrol as shown in FIG. 18B.

FIG. 19 shows that the NC_(Ca-ATP) channel in an R1 astrocyte from amale is inhibited by estrogen. The initial portion of the record showsbrisk activity from a number of superimposed channels, recorded in acell attached patch of membrane from an R1 astrocyte obtained from amale. Addition of 10 nM estrogen to the bath promptly resulted in stronginhibition of channel activity.

FIGS. 20A-20D show the gliotic capsule. FIG. 20A shows a coronal sectionof a rat brain sectioned though the site of implantation of a largegelatin sponge; the sponge (innermost dark region) is encapsulated by agliotic capsule (light area), outside of which is found a region ofvasogenic edema (outer dark area), identified by pre-mortemadministration of methylene blue. FIGS. 20B and 20C show low power andhigh power views, respectively, of the gliotic capsule immunolabeled forGFAP. FIG. 20D shows a high power view of GFAP-labeled cells inside ofthe gelatin sponge implant.

FIGS. 21A-21H show immunolabeled astrocytes. FIGS. 21A, 21C, 21E showfreshly-isolated large phase-bright R1 astrocytes immunolabeled for GFAP(FIG. 21C) and vimentin (FIG. 21E). FIGS. 21B, 21D, and 21F showfreshly-isolated small phase-dark R2 astrocytes immunolabeled for GFAP(FIG. 21D) and vimentin (FIG. 21F). FIG. 21G shows primary cultures ofastrocytes isolated from a gliotic capsule, with R1 astrocytesdeveloping into large polygonal cells (b), and R2 astrocytes developinginto small bipolar cells (a). FIG. 21H shows that R2 astrocytes, but notR1 astrocytes, are labeled with fluorescein tagged chlorotoxin derivedfrom the scorpion, Leiurus quinquestriatus.

FIGS. 22A-22D show that the inner zone of the gliotic capsule expressesSUR1 but not SUR2 Immunolabling for SUR1 (FIG. 22A) showed prominentexpression in cells adjacent to the gelatin sponge (gf), whereasimmunolabeling for SUR2 showed no expression (FIG. 22B). A single cellenzymatically isolated from a gelatin sponge implant and immunolabeledfor SUR1 is shown (FIG. 22C). FIG. 22D shown RT-PCR for SUR1 in controlinsulinoma cells (lane 2) and in isolated R1 astrocytes (lane 3), andfor SUR2 in control cardiac cells (lane 4), but not in isolated R1astrocytes (lane 5).

FIGS. 23A-23I show various features of the gliotic capsule. The glioticcapsule is characterized by GFAP-positive cells that are severalcell-layers thick (FIG. 23A). Only the inner zone of the gliotic capsuleis hypoxic, as demonstrated by pimonidozole labeling (FIG. 23B) and byimmunolabeling for HIF1α (FIG. 23C). Also, only the inner zone isimmunolabeled for SUR1 (FIG. 23D), and for the tight junction proteins,ZO-1 (FIG. 23E) and occludens (FIG. 23F). FIGS. 23G-23I show thatpimonidazole, HIF1α and occludens all localize to GFAP-positiveastrocytes that form the inner zone of the gliotic capsule.

FIGS. 24A and 24B show effects of NC_(Ca-ATP) channel inhibition (FIG.24A) and NC_(Ca-ATP) channel activation (FIG. 24B) on the glioticcapsule. Animals with gelatin sponge implants were treated withglibenclamide infusion (FIG. 24A) or diazoxide infusion (FIG. 24B) viaosmotic mini-pumps that delivered the compounds directly into the areaof the gelatin sponge. Immunolabeling for GFAP showed that channelinhibition with glibenclamide resulted in formation of a well definedgliotic capsule (FIG. 24A), whereas channel activation with diazoxideresulted in formation of a broader, ill-defined capsule (FIG. 24B), dueto diazoxide-induced necrotic death of inner zone cells.

FIGS. 25A and 25B show that infusion of diazoxide into the area aroundthe gelatin sponge resulted in a heavy infiltration of polymorphonuclearleukocytes (PMNs). Nuclear labeling with DAPI showed densely packedsmall cells in the vicinity of the gelatin sponge (FIG. 25A), withimmunolabeling using the PMN-specific marker, MMP-8, demonstrating thatthese cells were PMNs (FIG. 25B). It is believed that the stronginflammatory response represented by the infiltrating PMNs was due todisruption of the barrier between brain and foreign body (gelatinsponge) normally formed by the inner zone of the gliotic capsule.

FIGS. 26A-26L show that R1 astrocytes in the inner zone of the glioticcapsule typically express SUR1, a marker for the NC_(Ca-ATP) channel.The inner zones of the gliotic capsules in rats with gelatin spongeimplants (FIGS. 26A-26C), in rats with cerebral abscess (FIGS. 26D-26F),and in humans with metastatic tumor (FIGS. 26J-26L) are shown. Alsoshown is the area of reactive gloss adjacent to a stroke in the rat(FIGS. 26G-26I) resulting from occlusion of the middle cerebral artery.In all cases, a field of cells is labeled for GFAP and co-labelled forSUR1, as indicated. Examples of single cells at high power are alsoshown for each condition.

FIGS. 27A-27C shows that stellate astrocytes near the edge of a strokeup-regulate SUR1 (FIG. 27A), a marker of the NC_(Ca-ATP) channel. In themiddle of the stroke, cells with altered morphology including blebbingare also immunolabeled for SUR1 (FIGS. 27B and 27C).

FIGS. 28A-28C show that glibenclamide protects from Na azide-inducedchannel opening and necrotic cell death. FIG. 28A shows phase contrastimages of 4 different freshly isolated R1 astrocytes observed over thecourse of 30 min each. The cell exposed to vehicle solution aloneremained phase bright with no pathological deterioration (control). Thecell depleted of ATP by exposure to Na azide (1 mM) developedprogressive blebbing consistent with cytotoxic edema. Similarly, thecell exposed to the NC_(Ca-ATP) channel opener, diazoxide, developedprogressive blebbing consistent with cytotoxic edema. The cell exposedto Na azide in the presence of glibenclamide remained phase bright withno pathological deterioration. FIGS. 28B and 28C show cell death ofisolated R1 astrocytes induced by ATP depletion in vitro. Freshlyisolated R1 astrocytes were labeled for necrotic death with propidiumiodide (PI) (FIG. 28B), or for apoptotic death with annexin V (FIG.28C), under control conditions, after exposure to Na azide (1 mM), orafter exposure to Na azide in the presence of glibenclamide (1 μM).Exposure to Na azide resulted mostly in necrotic death that was largelyprevented by glibenclamide.

FIGS. 29A-29L shows that SUR1 is up-regulated in MCA stroke. Watershedarea between MCA-ACA in 3 different animals 8-16 hr after MCA stroke,identified by pre-mortem administration of Evans blue and postmortemperfusion with India ink (FIG. 29A), by TTC staining (FIG. 29B) and byimmunofluorescence imaging for SUR1 (FIG. 29C). Immunofluorescenceimages showing SUR1 at 3 hr in the core of the stroke in cells (FIG.29D) double-labeled for the neuronal marker, NeuN (FIG. 29E), andshowing SUR1 at 8 hr in the peri-infarct region in cells (FIGS. 14G and14J) double-labeled for the astrocytic marker, GFAP (FIG. 29H), and theendothelial cell marker, von Willebrand factor (FIG. 29K). Superimposedimages of double-labeled fields are shown (FIGS. 29F, 29I, and 29L).

FIGS. 30A-30G show that SUR1 but not Kir6.1 or Kir6.2 istranscriptionally up-regulated in MCA stroke. Western blots for SUR1(480 kDa) at different times (FIG. 30A) and in different locations (FIG.15B) after MCA stroke; in (FIG. 30A), lysates were all from TTC(+)peri-infarct regions of the involved hemisphere, obtained at the timesindicated; in (FIG. 30B), lysates were all obtained 8 hr after MCA stokefrom the regions indicated; each individual lane in a and b is from asingle animal. Quantification of the data from (FIG. 30A) and (FIG.30B), respectively, combined with comparable data for Kir6.1 and Kir6.2;for each individual blot, data were normalized to values of β-actin andto the control data for that blot and analyzed separately; **, p<0.01.In situ hybridization for SUR1, 3 hr after MCA stroke; paraffin sectionsshowed that large neuron-like cells (FIG. 30E) and capillaries (FIG.30F) in the ischemic zone were labeled, whereas tissues from the sameareas on the control side were not (FIG. 30G).

FIGS. 31A-31D show patch clamp recordings of NC_(CaATP) channel inneuron-like cells in stroke. FIG. 31A shows phase-contrast image oflarge neuron-like cells enzymatically isolated from ischemic region 3 hrfollowing MCAO. FIG. 31B shows recording of inside-out patch using Cs⁺as the charge carrier; channel activity was blocked by glibenclamidegiven as indicated (arrow); a and b show expanded records of theportions indicated. FIG. 31C shows recordings at potentials indicated ofinside-out patch using K⁺ as the charge carrier; channel activity wasblocked by glibenclamide. FIG. 31D shows a plot of single channelamplitudes at different voltages showing single channel slopeconductance of 34 pS.

FIGS. 32A-32E show that glibenclamide reduces mortality, edema andstroke size in MCA stroke. In FIG. 32A, Mortality was assessed during 7days after MCA stroke [double occlusion model with malignant cerebraledema (MCE)] in two treatment groups, each comprised of 19 female and 10male rats, treated with either saline (empty symbols) or glibenclamide(filled symbols); mortality at 7 days was significantly different.Subgroup analyses for males and females showed similar results. In FIG.32B edema was assessed 8 hr after MCA stroke (MCE model) in twotreatment groups, each comprised of 6 male rats treated with eithersaline or glibenclamide; tissues were first processed with TTC to allowseparation into TTC(+) and TTC(−) portions of the involved hemisphereand contralateral hemisphere, prior to determining wet/dry weights;values in TTC(+) regions were statistically different. In FIGS. 32C-32E,stroke size was assessed 48 hr after MCA stroke [thromboembolic (TE)model] in two treatment groups, each comprised of 10 male rats, treatedwith either saline or glibenclamide; images of TTC-stained coronalsections following MCA stroke (TE model) in an animal treated withsaline (FIG. 32C) and another treated with glibenclamide (FIG. 32D),showing cortical sparing often associated with glibenclamide treatment;values of stroke size, expressed as percent of hemisphere volume (FIG.32E).

FIGS. 33A-33D show that tissue distribution of BODIPY-glibenclamide inMCA stroke. a-c, Fluorescence images of brain sections in an animal 6 hrafter MCA stroke (MCE model) and administration of BODIPY-glibenclamide;fluorescent labeling was evident in cells, microvessels (FIG. 33A) andcapillaries (FIG. 33C) from ischemic regions, but not in thecontralateral hemisphere (FIG. 18B); the images in (FIGS. 33A and 33B)are from the same animal, taken with the same exposure time; in (FIG.33C), the single layer of nuclei confirms that the structure brightlylabeled by BODIPY-glibenclamide is a capillary. In FIG. 33D,immunofluorescence image of a brain section from an animal 6 hr afterMCA stroke (MCE model) labeled with anti-SUR1 antibody showing stronglabeling in a capillary and in adjacent neuron-like cells.

FIGS. 34A-34H show that glibenclamide reduces hemorrhagic conversion.FIGS. 34A-34D are from animals co-treated with saline; FIGS. 34E-34H arefrom animals co-treated with glibenclamide. The left column ofphotographs of coronal sections shows, in rows 1-2 only,intraventricular hemorrhage, plus large areas of hemorrhagic conversionin ischemic cortical/subcortical regions (red areas on the right side ofpictures; arrows). The right column of photographs of TTC-processedsections from the same animals show the areas of infarction.

FIGS. 35A and 35B show zymography showing gelatinase activity of matrixmetalloproteinases (MMP's) in stroke, and absence of direct MMPinhibition by glibenclamide. FIG. 35A shows activation of MMP-9 & MMP-2in stroke tissue compared to control; activity of recombinant MMP-9 &MMP-2 shown at left. FIG. 35B shows gelatinase activity of recombinantenzyme and stroke tissue under control conditions (CTR), in presence ofglibenclamide (10 μM), and in presence of MMP inhibitor II (300 nM;Calbiochem).

FIG. 36 shows phase contrast photomicrograph of cerebral capillariesfreshly isolated from normal brain, after enzymatic cleaning inpreparation for patch clamping.

FIGS. 37A-37F show that freshly isolated cerebral endothelial and smoothmuscle cells are readily distinguished electrophysiologically. FIGS. 37Aand 37B show superimposed macroscopic currents recorded during 200 msdepolarizing pulses from −120 mV to +120 mV in 20 mV steps in anendothelial cell (FIG. 37A) and in an elongated smooth muscle cell (FIG.37B); holding potential, −60 mV; nystatin perforated patch technique;bath solution, standard Krebs with 2 mM Ca²⁺; pipette solution, 145 mMK⁺. FIGS. 37C and 37D show current-voltage curves computed from average(mean±SE) currents at the end of 200-ms test pulses recorded in 9endothelial cells (FIG. 37C) and 7 smooth muscle cells (FIG. 37D); sameholding potential, technique and solutions as in FIGS. 37A and 37B.FIGS. 37E and 37F show current voltage curves recorded during ramppulses (0.45 mV/ms, holding potential, −60 mV) in an endothelial cell(FIG. 37E) and in a smooth muscle cell (FIG. 37F); same holdingpotential, technique and bath solution as in FIGS. 37A and 37B, but withpipette solution containing 145 mM Cs⁺ instead of K⁺.

FIG. 38 shows real time RT-PCR showing up-regulation of SUR1-mRNA instroke.

FIGS. 39A-39E show SUR1 knock down (SUR1KD) in R1 astrocytes protectsfrom ATP-depletion-induced depolarization. FIGS. 39A and 39B showWestern blot (FIG. 39A) and quantification of Western blots (FIG. 39B)of R1 cell lysates confirmed knock down of SUR1 expression by antisense.FIGS. 39C-39E show Na azide caused large depolarizations in cellsexposed to SCR-ODN (FIGS. 39C and 39E) but little or no depolarizationin cells exposed to AS-ODN (FIGS. 39D and 39E).

FIGS. 40A-40F show transcription factors in stroke. Immunofluorescenceimages of subcortical watershed region between ACA and MCA territories,from ipsilateral pen-infarct tissues 8 hr after MCAO (FIGS. 40A-D) andfrom contralateral control tissues (FIGS. 40E and 40F). The peri-infarctregion showed up-regulation of both transcription factors, Sp1 (FIGS.40A and 40C) and HIF1α (FIG. 40B) in neuron-like cells and capillaries,as well as SUR1 in capillaries (FIG. 40D). Control tissues showed littleSP1 and no HIF1α (FIGS. 40E and 40F).

FIGS. 41A-41C show an increase in nuclear localization of thetranscription factor, SP1, and SP1 co-localization with SUR1 in strokeImmunofluorescence images showing increase of nuclear SP1 labeling inischemic area 3-hr after MCAO (FIG. 41B), compared to contralateral side(FIG. 41A). FIG. 41C double labeling of large neuron-like cell showingnuclear SP1 (green) and cytoplasmic/plasmalemmal SUR1 (red) in the samecell.

FIGS. 42A-42D show regulation of SUR1 expression by the transcriptionfactor, HIF1α. FIGS. 42A and 42C show Western blot analysis of HIF1αprotein in R1 astrocytes from gelfoam implant model of control (CTR) andHIF1α knock-down (KD). FIGS. 42B and 42C show SUR1 protein in the samecell lysates.

FIG. 43 shows relative cerebral blood flow, measured by Laser DopplerFlowmetry, before (CTR), 1 hr after and 48 hr after MCAO, in 2 groups,each consisting of 4 male rats, treated with either saline orglibenclamide; values at 48 hr were statistically different (by ANOVA;p<0.01).

FIG. 44 Glibenclamide was just as effective in reducing edema afterstroke with added glucose as without added glucose. Supplemental glucose(1 gm/kg, i.p.) was administered 4 hr after MCAO, and animals weresacrificed 8 hr after MCAO for measurements of edema.

FIG. 45 Glibenclamide reduces stroke volume even when administration isdelayed up to 2 hours (low dose) or up to 6 hours (higher dose)following stroke.

FIG. 46 Glibenclamide reduces hemorrhagic conversion. Animals treatedwith intravenous tPA (10 mg/kg over 30 min) following thromboemboliclesion were also treated with either saline or glibenclamide. Although 5of 6 animals co-treated with saline showed hemorrhagic conversion, only1 of 6 animals treated with glibenclamide showed hemorrhagic conversion,demonstrating the efficacy of glibenclamide treatment to reduce orprevent hemorrhagic conversion following thromboembolic stroke.

FIG. 47 shows expression of SUR1 protein in cortical brain tissues;minimal labeling was observed in control tissues (left panel), whereasprominent labeling was seen surrounding the site of the impact (“I”,originating from the right side), in brain contusion (right panel);tissues were harvested 24 hr following contusion injury.

FIG. 48 shows high power views of previous image (above/right panel),showing SUR1 expression following brain contusion; SUR1 expression wasseen in large neuron-like cells (left panel) and in capillariesco-labeled with SUR1 and von Willebrand factor (middle and rightpanels).

DETAILED DESCRIPTION

The present invention relates to a novel ion channel whose functionunderlies the swelling of mammalian neural cells, such as in response toATP depletion; treatment methods related to diseases, trauma, andconditions that lead to the expression of such channels, including theuse of inhibitors of the channel function to prevent this cell swellingresponse, which characterizes brain damage in cerebral ischemia andtraumatic brain injury. The present invention also relates to the use ofthe channel to screen for channel inhibitors and activators, and otheruses.

The NC_(Ca-ATP) channel of the present invention is distinguished bycertain functional characteristics, the combination of whichdistinguishes it from known ion channels. The characteristics thatdistinguish the NC_(Ca-ATP) channel of the present invention include,but are not necessarily limited to, the following: 1) it is anon-selective cation channel that readily allows passage of Na, K andother monovalent cations; 2) it is activated by an increase inintracellular calcium, and/or by a decrease in intracellular ATP; 3) itis regulated by sulfonylurea receptor type 1 (SURD, which heretofore hadbeen considered to be associated exclusively with K_(ATP) channels suchas those found in pancreatic β cells, for example.

More specifically, the NC_(Ca-ATP) channel of the present invention hasa single-channel conductance to potassium ion (K⁺) between 20 and 50 pS.The NC_(Ca-ATP) channel is also stimulated by Ca²⁺ on the cytoplasmicside of the cell membrane in a physiological concentration range, wheresaid concentration range is from 10⁻⁸ to 10⁻⁵ M. The NC_(Ca-ATP) channelis also inhibited by cytoplasmic ATP in a physiological concentrationrange, where said concentration range is from about 10⁻¹ to about 10 μM.The NC_(Ca-ATP) channel is also permeable to the following cations; K⁺,Cs⁺, Li⁺, Na⁺; to the extent that the permeability ratio between any twoof said cations is greater than 0.5 and less than 2.

Some of the preferred embodiments of the present invention will bedescribed in detail with reference to the attached drawings.

This invention may be embodied in many different forms and should not beconstrued as being limited to the embodiments set forth herein.

I. NC_(Ca-ATP) CHANNEL

A unique non-selective monovalent cationic ATP-sensitive channel(NC_(Ca-ATP) channel) was identified first in native reactive astrocytes(NRAs) and later, as described herein, in neurons and capillaryendothelial cells after stroke or traumatic brain or spinal cord injury(See at least International application WO 03/079987 to Simard et al.,and Chen and Simard, 2001, each incorporated by reference herein in itsentirety). As with the K_(ATP) channel in pancreatic β cells, theNC_(CaATP) channel is thought to be a heteromultimer structure comprisedof sulfonylurea receptor type 1 (SUR1) regulatory subunits andpore-forming subunits (Chen et al., 2003). The pore-forming subunitshave been characterized biophysically, but have yet to be characterizedmolecularly.

The invention is based, in part, on the discovery of a specific channel,the NC_(Ca-ATP) channel, defined as a channel on astrocytes in USApplication Publication No. 20030215889, which is incorporated herein byreference in its entirety. More specifically, the present invention hasfurther defined that this channel is not only expressed on astrocytes,it is expressed at least on neural cells, neuroglial cells, and/orneural endothelial cells after brain and spinal cord trauma, forexample, an hypoxic event, an ischemic event, or other secondaryneuronal injuries relating to these events.

The NC_(Ca-ATP) channel is activated by calcium ions (Ca²⁺) and issensitive to ATP. Thus, this channel is a non-selective cation channelactivated by intracellular Ca²⁺ and blocked by intracellular ATP. Whenopened by depletion of intracellular ATP, this channel is responsiblefor complete depolarization due to massive Na⁺ influx, which creates anelectrical gradient for Cl⁻ and an osmotic gradient for H₂O, resultingin cytotoxic edema and cell death. When the channel is blocked orinhibited, massive Na⁺ does not occur, thereby preventing cytotoxicedema.

Certain functional characteristics distinguish the NC_(Ca-ATP) channelfrom other known ion channels. These characteristics can include, butare not limited to, at least some of the following: 1) it is anon-selective cation channel that readily allows passage of Na⁺, K⁺ andother monovalent cations; 2) it is activated by an increase inintracellular calcium, and/or by a decrease in intracellular ATP; 3) itis regulated by sulfonylurea receptor type 1 (SUR1), which heretoforehad been considered to be associated exclusively with K_(ATP) channelssuch as those found in pancreatic β cells.

More specifically, the NC_(Ca-ATP) channel of the present invention hasa single-channel conductance to potassium ion (K⁺) between 20 and 50 pS.The NC_(Ca-ATP) channel is also stimulated by Ca²⁺ on the cytoplasmicside of the cell membrane in a physiological concentration range, whereconcentration range is from 10⁻⁸ to 10⁻⁵ M. The NC_(Ca-ATP) channel isalso inhibited by cytoplasmic ATP in a physiological concentrationrange, where the concentration range is from 10⁻¹ to 10 M. TheNC_(Ca-ATP) channel is also permeable to the following cations; K⁺, Cs⁺,Li⁺, Na⁺; to the extent that the permeability ratio between any two ofthe cations is greater than 0.5 and less than 2.

SUR imparts sensitivity to antidiabetic sulfonylureas such asglibenclamide and tolbutamide and is responsible for activation by achemically diverse group of agents termed “K⁺ channel openers” such asdiazoxide, pinacidil and cromakalin (Aguilar-Bryan et al., 1995; Inagakiet al., 1996; Isomoto et al., 1996; Nichols et al., 1996; Shyng et al.,1997). In various tissues, molecularly distinct SURs are coupled todistinct pore-forming subunits to form different K_(ATP) channels withdistinguishable physiological and pharmacological characteristics. TheK_(ATP) channel in pancreatic β cells is formed from SUR1 linked withKir6.2, whereas the cardiac and smooth muscle K_(ATP) channels areformed from SUR2A and SUR2B linked with Kir6.2 and Kir6.1, respectively(Fujita et al., 2000). Despite being made up of distinctly differentpore-forming subunits, the NC_(Ca-ATP) channel is also sensitive tosulfonylurea compounds.

Also, unlike the K_(ATP) channel, the NC_(Ca-ATP) channel conductssodium ions, potassium ions, cesium ions and other monovalent cationswith near equal facility (Chen and Simard, 2001) suggesting further thatthe characterization, and consequently the affinity to certaincompounds, of the NC_(Ca-ATP) channel differs from the K_(ATP) channel.

Other nonselective cation channels that are activated by intracellularCa²⁺ and inhibited by intracellular ATP have been identified by othersbut not in astrocytes or neurons as disclosed herein. Further, theNC_(Ca-ATP) channel expressed and found in astrocytes differsphysiologically from the other channels with respect to calciumsensitivity and adenine nucleotide sensitivity (Chen et al., 2001).

Summary of NC_(Ca-ATP) Channel Characteristics

At least some of the characteristics of cells expressing and compositioncomprising the NC_(Ca-ATP) channel of the present invention aresummarized in Table 1 (taken from experiments with freshly isolatednative reactive astrocytes [NRA]).

TABLE 1 Properties of cells and membrane compositions containing theNC_(Ca-ATP) Channel of the Present Invention Membrane Preparationderived from freshly isolated native Reactive Astrocytes reactiveastrocytes Monovalent cation Yes: Yes: permeable? Na⁺ Na⁺ K⁺ K⁺ Li⁺ Li⁺Rb⁺ Rb⁺ Cs⁺ Cs⁺ (Na⁺ ≈ K⁺ ≈ (NA⁺ ≈ K⁺ ≈ Li⁺ ≈ Rb⁺) Li⁺ ≈ Rb⁺) Anionpermeable? No No Divalent cation No No permeable? Compounds blockingSUR1 antagonists SUR1 ANTAGONISTS channel activity Channel openingIntracell. ATP Intracell ATP Requires: depletion depletion Intracell.Mg²⁺ Intracell. Mg²⁺ Single Channel ~35 pS ~35 PS Conductance Activation<1.0 μM <1.0 μM [Ca²⁺] [ATP]₁ EC₅₀ (um) 0.79 μM 0.79 μM ADP No channeleffect No channel effect AMP Pore radius 0.41 0.41 (mm)

II. GLIOTIC CAPSULE

The gliotic capsule forms a potentially harmful mass of tissue thatcontributes to brain swelling and mass effect, and that may shelterforeign cells from surveillance by the immune system. Applicants are thefirst to determine that, in a variety pathological conditions in bothrats and humans, reactive astrocytes (R1 astrocytes) in the inner zoneof the gliotic capsule express a novel SUR1-regulated cation channel,the NC_(Ca-ATP) channel, and that this channel directly controls cellviability: opening the channel is associated with necrotic cell deathand closing the channel is associated with protection from cell deathinduced by energy (ATP) depletion.

As described herein, Applicants are the first to determine that theinner zone of the gliotic capsule is populated by R1 astrocytesexpressing the NC_(Ca-ATP) channel. Selectively killing the astrocytesexpressing the NC_(Ca-ATP) channel may aid in the treatment ofconditions that lead to the formation of gliotic capsules. For example,selectively killing the astrocytes expressing the NC_(Ca-ATP) channeldisrupts the “tumor brain barrier” (TBB), causing migration ofleukocytes across the TBB and aiding in treatment of tumors in thebrain.

Also there exists a need for therapeutic compounds capable of modulatingthe activity of this target in order to prevent brain damage. Thepresent invention is directed to a newly characterized non-selectivecalcium and ATP sensitive monovalent cation channel, termed theNC_(Ca-ATP) channel, which is present in neural cells and linked to anSUR. The present invention further provides a method to screen for oridentify antagonists to NC_(Ca-ATP) channel activity. Further, thepresent invention provides a method for the therapeutic use ofantagonists, such as sulfonylureas and other SUR1 blockers, to inhibitthis channel's activity and thereby prevent neural cell swelling andcell death and the concomitant nervous system damage that includes brainswelling and brain damage.

Sodium azide (NaN₃) is a metabolic toxin used to induce “chemicalhypoxia” by depleting intracellular ATP. See, Swanson, 1992. Themorphological and electrophysiological responses of neural cells to NaN₃are examined in a novel cell preparation. Freshly isolated nativereactive astrocytes (NRAs) from adult rat brain are used and studied ina native state immediately after their isolation. Reactive astrocytesare astrocytes that have been activated or stimulated in vivo, such asthose associated with brain or neural injury. In the post-mortem brainsof traumatic brain injury (TBI) patients, reactive astrocytes are foundin proximity to the injury. The majority of reactive astrocytessurrounding an injury site in the brain are reactive astrocytes. Type 1reactive astrocytes comprise >80% of recoverable reactive astrocytes,whereas type 2 reactive astrocytes comprise about 5%. Reactiveastrocytes are normally polarized under quiescent conditions.

It is readily apparent to one skilled in the art that variousembodiments and modifications can be made to the invention disclosed inthis application without departing from the scope and spirit of theinvention.

III. DEFINITIONS

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” Some embodiments of the invention mayconsist of or consist essentially of one or more elements, method steps,and/or methods of the invention. It is contemplated that any method orcomposition described herein can be implemented with respect to anyother method or composition described herein.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativeare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used herein, the term “acute” refers to the onset of a health effect,usually the effect is a rapid onset that is considered brief, notprolonged.

As used herein, the term “acute cerebral ischemia” refers to a cerebralischemic event that has a rapid onset and is not prolonged. The terms“acute cerebral ischemia” and “stroke” can be used interchangeably.”

As used herein, the term “agonist” refers to a biological or chemicalagent that combines with a receptor on a cell and initiates the same orequivalent reaction or activity produced by the binding of an endogenoussubstance. In the present invention, the agonist combines, binds, and/orassociates with a NC_(Ca-ATP) channel of a neuronal cell, a neuroglialcell, or a neural endothelial cell, such that the NC_(Ca-ATP) channel isopened (activated). In certain embodiments, the agonist combines, bindsand/or associates with a regulatory subunit of the NC_(Ca-ATP) channel,particularly a SUR1. Alternatively, the agonist combines, binds, and/orassociates with a pore-forming subunit of the NC_(Ca-ATP) channel, suchthat the NC_(Ca-ATP) channel is opened (activated). The terms agonistand/or activator can be used interchangeably.

As used herein, the term “antagonist” refers to a biological or chemicalagent that acts within the body to reduce the physiological activity ofanother chemical or biological substance. In the present invention, theantagonist blocks, inhibits, reduces and/or decreases the activity of aNC_(Ca-ATP) channel of a neuronal cell, a neuroglia cell or a neuralendothelial cell (e.g., capillary endothelial cells). In the presentinvention, the antagonist combines, binds, associates with a NC_(Ca-ATP)channel of neuronal cell, a neuroglia cell or a neural endothelial cell(e.g., capillary endothelial cells), such that the NC_(Ca-ATP) channelis closed (deactivated), meaning reduced biological activity withrespect to the biological activity in the diseased state. In certainembodiments, the antagonist combines, binds and/or associates with aregulatory subunit of the NC_(Ca-ATP) channel, particularly a SUR1.Alternatively, the antagonist combines, binds, and/or associates with apore-forming subunit of the NC_(Ca-ATP) channel, such that theNC_(Ca-ATP) channel is closed (deactivated). The terms antagonist orinhibitor can be used interchangeably.

As used herein, the terms “brain abscess” or “cerebral abscess” refer toa circumscribed collection of purulent exudate that is typicallyassociated with swelling.

As used herein, the terms “blood brain barrier” or “BBB” refer thebarrier between brain blood vessels and brain tissues whose effect is torestrict what may pass from the blood into the brain.

As used herein, the term “cerebral ischemia” refers to a lack ofadequate blood flow to an area, for example a lack of adequate bloodflow to the brain or spinal cord, which may be the result of a bloodclot, blood vessel constriction, a hemorrhage or tissue compression froman expanding mass.

As used herein, the term “depolarization” refers to an increase in thepermeability of the cell membrane to sodium ions wherein the electricalpotential difference across the cell membrane is reduced or eliminated.

As used herein, the terms “effective amount” or “therapeuticallyeffective amount” are interchangeable and refer to an amount thatresults in an improvement or remediation of the symptoms of the diseaseor condition. Those of skill in the art understand that the effectiveamount may improve the patient's or subject's condition, but may not bea complete cure of the disease and/or condition.

As used herein, the term “endothelium” refers a layer of cells that linethe inside surfaces of body cavities, blood vessels, and lymph vesselsor that form capillaries.

As used herein, the term “endothelial cell” refers to a cell of theendothelium or a cell that lines the surfaces of body cavities, forexample, blood or lymph vessels or capillaries. In certain embodiments,the term endothelial cell refers to a neural endothelial cell or anendothelial cell that is part of the nervous system, for example thecentral nervous system or the brain or spinal cord.

As used herein, the term “gliotic capsule” refers to a physical barriersurrounding, in whole or in part, a foreign body, including a metastatictumor, a cerebral abscess or other mass not normally found in brainexcept under pathological conditions. In certain embodiments, thegliotic capsule comprises an inner zone comprising neuronal cells,neuroglial cells (e.g., astrocytes) and/or endothelial cells expressinga NC_(Ca-ATP) channel.

As used herein, the term “ionic edema” in brain or nervous tissue refersto edema arising in tissue in which the blood-brain barrier remainssubstantially intact, and is associated with the movement ofelectrolytes (e.g. Na⁺, Cl⁻) plus water into brain parenchyma.

As used herein, the term “inhibit” refers to the ability of the compoundto block, partially block, interfere, decrease, reduce or deactivate achannel such as the NC_(Ca-ATP) channel. Thus, one of skill in the artunderstands that the term inhibit encompasses a complete and/or partialloss of activity of a channel, such as the NC_(Ca-ATP) channel. Channelactivity may be inhibited by channel block (occlusion or closure of thepore region, preventing ionic current flow through the channel), bychanges in an opening rate or in the mean open time, changes in aclosing rate or in the mean closed time, or by other means. For example,a complete and/or partial loss of activity of the NC_(Ca-ATP) channel asmay be indicated by a reduction in cell depolarization, reduction insodium ion influx or any other monovalent ion influx, reduction in aninflux of water, reduction in extravasation of blood, reduction in celldeath, as well as an improvement in cellular survival following anischemic challenge.

The term “morbidity” as used herein is the state of being diseased. Yetfurther, morbidity can also refer to the disease rate or the ratio ofsick subjects or cases of disease in to a given population.

The term “mortality” as used herein is the state of being mortal orcausing death. Yet further, mortality can also refer to the death rateor the ratio of number of deaths to a given population.

As used herein, the term “neuron” refers to a nerve cell, also termed aneuronal cell.

As used herein, the term “neuronal cell” refers to a cell that is amorphologic and functional unit of the nervous system. The cellcomprises a nerve cell body, the dendrites, and the axon. The termsneuron, nerve cell, neuronal, neurone, and neurocyte can be usedinterchangeably. Neuronal cell types can include, but are not limited toa typical nerve cell body showing internal structure, a horizontal cell(of Cajal) from cerebral cortex; Martinottic cell, biopolar cell,unipolar cell, Pukinje cell, and a pyramidal cell of motor area ofcerebral cortex.

As used herein, the term “neural” refers to anything associated with thenervous system. As used herein, the term “neural cells” includes neuronsand glia, including astrocytes. As used herein, the term “isolatedneural cells” means neural cells isolated from brain.

As used herein, the terms “neuroglia” or “neuroglial cell” refers to acell that is a non-neuronal cellular element of the nervous system. Theterms neuroglia, neurogliacyte, and neuroglial cell can be usedinterchangeably. Neuroglial cells can include, but are not limited toependymal cells, astrocytes, oligodendrocytes, or microglia.

The term “preventing” as used herein refers to minimizing, reducing orsuppressing the risk of developing a disease state or parametersrelating to the disease state or progression or other abnormal ordeleterious conditions.

The term “reactive astrocytes” means astrocytes found in brain at thesite of a lesion or ischemia. The term “native reactive astrocytes” or“NRAs” means reactive astrocytes that are freshly isolated from brain.The term “freshly isolated” as used herein refers to NRAs that have beenpurified from brain, particularly NRAs that were purified from about 0to about 72 hours previously. When NRAs are referred to as being“purified from brain” the word “purified” means that the NRAs areisolated from other brain tissue and/or implanted gelatin or sponge anddoes not refer to a process that simply harvests a population of cellsfrom brain without further isolation of the cells. As described herein,the NC_(Ca-ATP) channel found in reactive astrocytes is present only infreshly isolated cells; the NC_(Ca-ATP) channel is lost shortly afterculturing the cells under typical normoxic conditions. NRAs provide anin vitro model that is more similar to reactive astrocytes as they existin vivo in the brain, than astrocytes grown in culture. The terms“native” and “freshly isolated” are used synonymously.

As used herein, the term “reduces” refers to a decrease in cell death,inflammatory response, hemorrhagic conversion, extravasation of blood,etc. as compared to no treatment with the compound of the presentinvention. Thus, one of skill in the art is able to determine the scopeof the reduction of any of the symptoms and/or conditions associatedwith a spinal cord injury in which the subject has received thetreatment of the present invention compared to no treatment and/or whatwould otherwise have occurred without intervention.

As used herein, the term “stroke” refers to any acute, clinical eventrelated to the impairment of cerebral circulation. The terms “acutecerebral ischemia” and “stroke” can be used interchangeably.

The terms “treating” and “treatment” as used herein refer toadministering to a subject a therapeutically effective amount of acomposition so that the subject has an improvement in the disease orcondition. The improvement is any observable or measurable improvement.Thus, one of skill in the art realizes that a treatment may improve thepatient's condition, but may not be a complete cure of the disease.Treating may also comprise treating subjects at risk of developing adisease and/or condition.

As used herein, the term “vasogenic edema” in brain or nervous tissuerefers to edema arising in tissue in which the blood-brain barrier isnot substantially intact, and in which macromolecules plus water enterinto brain parenchyma in addition to any movement of electrolytes.

Reactive astrocytes are produced in vivo and harvested from brainaccording to a method system similar to that described by Perillan. See,Chen et al., 2003; Chen et al., 2001, for example. Harvested cells arethen isolated and not cultured; rather, the freshly isolated reactiveastrocytes are studied in a native state immediately after theirisolation from the brain. As described by Perillan et al. (1999; 2000),cultured astrocytes do not express the NC_(Ca-ATP) channel.

The Examples described herein reveal that NRAs from adult rat brainexpress a non-selective cation channel that is activated by depletion of[ATP]_(i) at physiological concentrations of [Ca²⁺]_(i). ThisNC_(Ca-ATP) channel of the present invention, which is newly identifiedin NRAs and present in >90% of membrane patches from such cells, isdistinguished from previously reported non-selective calcium and ATPchannels by exhibiting significantly different properties. Thesedistinguishing properties of the NC_(Ca-ATP) of the present inventioninclude: being activated by submicromolar [Ca²⁺] and exhibiting adifferent sensitivity to block by various adenine nucleotides. Openingof the NC_(Ca-ATP) channel of the present invention by ATP depletioncauses profound membrane depolarization, which precedes blebbing of thecell membrane. Upon ATP depletion, the NC_(Ca-ATP) channel opens toallow Na⁺ influx that leads to cell swelling. This channel is regulatedby sulfonylurea receptor type 1 (SUR1). The channel can be blocked bysulfonylurea, such as glibenclamide and tolbutamide; treatment withglibenclamide results in significant reduction in swelling and blebbingand cell death induced by chemical ATP depletion. This channelparticipates in the cation flux involved in cell swelling and celldeath. A method of the present invention includes the use ofsulfonylurea compounds to inhibit the flow of current through theNC_(Ca-ATP) channel and inhibit blebbing related to channel opening.Also, use of sulfonylurea compounds and other compounds that inhibit theflow of current through the NC_(Ca-ATP) channel, thus can have atherapeutic preventative effect on cell swelling and cell death in thebrain and spinal cord.

In some embodiments, the present invention is directed to therapeuticcompositions and methods of using the same. In one embodiment, thetherapeutic composition is an agonist and/or antagonist of at least oneNC_(Ca-ATP) channel of a neuronal cell, a neuroglial cell, or a neuralendothelial cell. Further embodiments of the present invention provide acomposition comprising a membrane preparation expressing the NC_(Ca-ATP)channel. For example, the membrane preparation is derived from neuralcells, such as isolated native reactive astrocytes (NRAs), preferablyfreshly isolated native reactive astrocytes. The NC_(Ca-ATP) channel inthe composition has the following characteristics: (a) it is a 35 pStype channel; (b) it is stimulated by cytoplasmic Ca²⁺; (c) it openswhen cytoplasmic ATP is less than about 0.8 μM; and (d) it is permeableto the monovalent cations K⁺, Cs⁺, Li⁺ and Na⁺ and it can be blocked byantagonists of the type 1 sulfonylurea receptor.

Furthermore, it is an object of the present invention to provide amethod of screening for one or more antagonists of the NC_(Ca-ATP)channel, comprising: (a) contacting a test compound with a compositioncomprising the NC_(Ca-ATP) channel; and (b) identifying test compoundsthat inhibit an activity of said channel by measuring said activity inthe presence and absence of said test compound, wherein a test compoundthat inhibits said activity is identified as an antagonist of theNC_(Ca-ATP) channel. For example, the composition may contain apreparation of neural cells expressing the NC_(Ca-ATP) channel or amembrane preparation expressing the NC_(Ca-ATP) channel, such as amembrane preparation derived from isolated native reactive astrocytes(NRAs) or other cells that express the NC_(Ca-ATP) channel. The effectof the compound on this channel may include: (a) blocking theNC_(Ca-ATP) channel; (b) closing the NC_(Ca-ATP) channel; (c) preventingthe NC_(Ca-ATP) channel from opening; and (d) reducing the magnitude ofmembrane current through the NC_(Ca-ATP) channel. It is also an objectof the present invention to identify a compound that is an NC_(Ca-ATP)antagonist, including an NC_(Ca-ATP) channel inhibitor, an NC_(Ca-ATP)channel blocker, a SUR1 antagonist, SUR1 inhibitor, and/or a compoundcapable of reducing the magnitude of membrane current though thechannel.

It is a further object of the invention to provide a method foridentifying compounds that inhibit neural cell swelling, comprising: (a)contacting a test compound with a composition comprising the NC_(Ca-ATP)channel, and (b) determining whether the test compound blocks theNC_(Ca-ATP) channel, wherein a test compound that blocks the NC_(Ca-ATP)channel is identified as a compound for inhibiting neural cell swelling.

It is a further object of the present invention to provide a method foridentifying compounds that inhibit brain swelling, comprising: (a)contacting a test compound with a composition comprising the NC_(Ca-ATP)channel, and (b) determining whether the test compound blocks theNC_(Ca-ATP) channel, wherein a test compound that blocks the NC_(Ca-ATP)channel is identified as a compound for inhibiting brain swelling.

Yet another object of the present invention is to provide a method foridentifying compounds that inhibit brain swelling, comprising: (a)contacting a test compound with a composition comprising the NC_(Ca-ATP)channel, and (b) determining whether the test compound inhibits neuralcell swelling, wherein a test compound that inhibits neural cellswelling is identified as a compound for inhibiting brain swelling.

A further object of the present invention provides a method foridentifying compounds that inhibit neural cell swelling in an animal,comprising: (a) contacting a test compound with a composition comprisingthe NC_(Ca-ATP) channel and determining whether the test compound blocksthe channel, and (b) administering the test compound to an animal havinga brain injury or cerebral ischemia, and determining whether the testcompound that inhibits brain swelling of the treated animal, whereintest compounds that inhibit brain swelling are identified as compoundsthat inhibit neural cell swelling in an animal.

It is a further object of the present invention to provide a method foridentifying compounds that inhibit brain swelling, comprising: (a)contacting a test compound with a composition comprising the NC_(Ca-ATP)channel, and determining whether the test compound blocks the channel,and (b) administering the test compound to an animal having a braininjury or cerebral ischemia, and determining whether the test compoundinhibits brain swelling of the treated animal, wherein test compoundsthat block the NC_(Ca-ATP) channel are identified as compounds thatinhibit brain swelling.

In each of these objects of the present invention, the compositionpreferably comprises a preparation of neural cells expressing theNC_(Ca-ATP) channel or a membrane preparation expressing the NC_(Ca-ATP)channel, which preferably is derived from isolated native reactiveastrocytes (NRAs). It is a further object of the present invention toprovide the above methods using a compound that is an antagonist of atype 1 sulfonylurea receptor, such as a sulfonylurea compound, abenzamido derivative or an imidazoline derivative.

It is a further object of the present invention to provide these methodsin which the determining step include, but are not limited to, detectingor identifying swelling of the native reactive astrocytes, such as bymicroscopic observation of cell appearance (normal, blebbing, swelling);measuring channel currents; measuring membrane potential; detectingexpression of annexin V; detecting expression of propidium iodide; invitro binding assays; and combinations thereof.

It is a further object of the present invention to provide a method ofpreventing neural cell swelling in the brain of a subject, said methodcomprising administering to the subject a formulation containing aneffective amount of a compound that blocks the NC_(Ca-ATP) channel and apharmaceutically acceptable carrier.

It is a further object of the present invention to provide a method ofalleviating the negative effects of traumatic brain injury or cerebralischemia stemming from neural cell swelling in a subject, comprisingadministering to the subject a formulation comprising an effectiveamount of a compound that blocks the NC_(Ca-ATP) channel and apharmaceutically acceptable carrier. Such administration may be deliverydirectly to the brain, intravenous, subcutaneous, intramuscular,intracutaneous, intragastric and oral administration. Examples of suchcompounds include antagonist of a type 1 sulfonylurea receptor, such assulfonylureas like glibenclamide and tolbutamide, as well as otherinsulin secretagogues such as repaglinide, nateglinide, meglitinide,midaglizole, LY397364, LY389382, gliclazide, glimepiride, MgADP, andcombinations thereof.

It is yet another object of the present invention to provide aformulation for preventing or inhibiting neural cell swelling in thebrain of a subject, using a formulation that includes a compound thatblocks the NC_(Ca-ATP) channel and a pharmaceutically acceptablecarrier, wherein the quantity of said compound is less than the quantityof said compound in formulations for treating diabetes. It is a furtherobject of the present invention to provide a formulation for preventingor inhibiting neural cell swelling in the brain of a subject, using aformulation that includes a compound that blocks the NC_(Ca-ATP) channeland a pharmaceutically acceptable carrier, wherein the quantity of saidcompound is at least 2 times less than the quantity of said compound informulations for treating diabetes. It is a further object of thepresent invention to provide a formulation for preventing or inhibitingneural cell swelling in the brain of a subject, using a formulation thatincludes a compound that blocks the NC_(Ca-ATP) channel and apharmaceutically acceptable carrier, wherein the quantity of saidcompound is at least 5 times less than the quantity of said compound informulations for treating diabetes. It is yet another object of thepresent invention to provide a formulation for preventing or inhibitingneural cell swelling in the brain of a subject, using a formulation thatincludes a compound that blocks the NC_(Ca-ATP) channel and apharmaceutically acceptable carrier, wherein the quantity of saidcompound is at least 10 times less than the quantity of said compound informulations for treating diabetes.

It is therefore another object of the present invention to provide amethod for identifying compounds that inhibit neural cell swelling,comprising: (a) contacting a test compound with a composition comprisingthe Kir2.3 channel, and (b) determining whether the test compound opensthe Kir2.3 channel, wherein a test compound that opens the Kir2.3channel is identified as a compound for inhibiting neural cell swelling.

It is yet another object of the present invention to provide a methodfor a method for identifying compounds that inhibit brain swelling,comprising: (a) contacting a test compound with a composition comprisingthe Kir2.3 channel, and (b) determining whether the test compound opensthe Kir2.3 channel, wherein a test compound that opens the Kir2.3channel is identified as a compound for inhibiting brain swelling.

It is yet another object of the present invention to provide a methodfor a method for identifying compounds that inhibit neural cell swellingand/or brain swelling in an animal, comprising: (a) contacting a testcompound with a composition comprising the Kir2.3 channel, and (b)determining whether the test compound opens the Kir2.3 channel, whereina test compound that opens the Kir2.3 channel is identified as acompound for inhibiting neural cell swelling and/or brain swelling in ananimal.

It is a further object of the present invention to provide a method foridentifying compounds that prevent, inhibit and/or alleviate brainswelling in a subject, comprising: (a) contacting a test compound with acomposition comprising the Kir2.3 channel, and determining whether thetest compound opens the Kir2.3 channel, and (b) administering the testcompound to a subject having a brain injury or cerebral ischemia, anddetermining whether the test compound prevents, inhibits and/oralleviates brain swelling in the subject, wherein test compounds thatopen the Kir2.3 channel are identified as compounds that inhibit brainswelling.

It is a further object of the present invention to provide a method foridentifying compounds that inhibit neural cell swelling in an animal,comprising: (a) contacting a test compound with a composition comprisingthe Kir2.3 channel, and determining whether the test compound opens theKir2.3 channel, and (b) administering the test compound to an animalhaving a brain injury or cerebral ischemia, and determining whether thetest compound inhibits brain swelling of the treated animal, whereintest compounds that inhibit brain swelling are identified as compoundsthat inhibit neural cell swelling in an animal.

It is also an object of the present invention to provide a method ofpreventing neural cell swelling in the brain of a subject, said methodcomprising administering to the subject a formulation containing aneffective amount of a compound that opens the Kir2.3 channel and apharmaceutically acceptable carrier.

It is a further objection of the present invention to provide a methodof alleviating the negative effects of traumatic brain injury orcerebral ischemia stemming from neural cell swelling in a subject,comprising administering to the subject a formulation comprising aneffective amount of a compound that opens the Kir2.3 channel and apharmaceutically acceptable carrier. In the object of the presentinvention that provide methods assessing the effect of a compound on theKir2.3 channel, a preferred compound is Tenidap(5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H-indole-1-carboxamide).For example the formulation may provide a daily dose of Tenidap that isfrom about 10 mg/day to about 500 mg/day, or, when administered directlyto the brain the daily dose of Tenidap is from about 500 mg/day to 1.5gms/day or greater.

IV. EXEMPLARY EMBODIMENTS OF THE PRESENT INVENTION

In addition to the sulfonylurea receptor 1 (SUR1) being expressed in R1astrocytes as part of the NC_(Ca-ATP) channel, the present inventionfurther describes that the SUR1 regulatory subunit of this channel isup-regulated in neurons and capillary endothelial cells followingischemia, and blocking this receptor reduces stroke size, cerebral edemaand mortality. Thus, antagonists of the NC_(Ca-ATP) channel may have animportant role in preventing, alleviating, inhibiting and/or abrogatingthe formation of cytotoxic and ionic edema.

In other embodiments, the therapeutic compound of the present inventioncomprises an antagonist of a NC_(Ca-ATP) channel of a neuronal cell, aneuroglial cell, a neural endothelial cell or a combination thereof.Antagonists are contemplated for use in treating adverse conditionsassociated with hypoxia and/or ischemia that result in increasedintracranial pressure and/or cytotoxic edema of the central nervoussystem. Such conditions include trauma, ischemic brain injury, namelysecondary neuronal injury, and hemorrhagic infarction. Antagonistsprotect the cells expressing the NC_(Ca-ATP) channel, which is desirablefor clinical treatment in which gliotic capsule integrity is importantand must be maintained to prevent the spread of infection, such as witha brain abscess. The protection via inhibition of the NC_(Ca-ATP)channel is associated with a reduction in cerebral edema.

In one aspect, the NC_(Ca-ATP) channel is blocked, inhibited, orotherwise is decreased in activity. In such examples, an antagonist ofthe NC_(Ca-ATP) channel is administered and/or applied. The antagonistmodulates the NC_(Ca-ATP) channel such that flux through the channel isreduced, ceased, decreased and/or stopped. The antagonist may have areversible or an irreversible activity with respect to the activity ofthe NC_(Ca-ATP) channel of the neuronal cell, neuroglial cell,endothelial cell or a combination thereof. The antagonist may prevent orlessen the depolarization of the cells thereby lessening cell swellingdue to osmotic changes that can result from depolarization of the cells.Thus, inhibition of the NC_(Ca-ATP) channel can reduce cytotoxic edemaand death of endothelial cells.

Subjects that can be treated with the therapeutic composition of thepresent invention include, but are not limited subjects suffering fromor at risk of developing conditions associated hypoxia and/or ischemiathat result in increased intracranial pressure and/or with cytotoxicedema of the central nervous system (CNS). Such conditions include, butare not limited to trauma (e.g., traumatic brain or spinal cord injury(TBI or SCI), concussion) ischemic brain injury, hemorrhagic infarction,stroke, atrial fibrillations, clotting disorders, pulmonary emboli,arterio-venous malformations, mass-occupying lesions (e.g., hematomas),etc. Still further subjects at risk of developing such conditions caninclude subjects undergoing treatments that increase the risk of stroke,for example, surgery (vascular or neurological), treatment of myocardialinfarction with thrombolytics, cerebral/endovascular treatments, stentplacements, angiography, etc.

Another aspect of the present invention for the treatment of ischemia,brain trauma, or other brain injury comprises administration of aneffective amount of a SUR1 antagonist and administration of glucose.Glucose administration may be at the time of treatment with anantagonist of the NC_(Ca-ATP) channel, such as a SUR1 antagonist, or mayfollow treatment with an antagonist of the NC_(Ca-ATP) channel (e.g., at15 minutes after treatment with an antagonist of the NC_(Ca-ATP)channel, or at one half hour after treatment with an antagonist of theNC_(Ca-ATP) channel, or at one hour after treatment with an antagonistof the NC_(Ca-ATP) channel, or at two hours after treatment with anantagonist of the NC_(Ca-ATP) channel, or at three hours after treatmentwith an antagonist of the NC_(Ca-ATP) channel). Glucose administrationmay be by intravenous, or intraperitoneal, or other suitable route andmeans of delivery. Additional glucose allows administration of higherdoses of an antagonist of the NC_(Ca-ATP) channel than might otherwisebe possible, so that combined glucose with an antagonist of theNC_(Ca-ATP) channel provides greater protection, and may allow treatmentat later times, than with an antagonist of the NC_(Ca-ATP) channelalone. Greater amounts of glucose are administered where larger doses ofan antagonist of the NC_(Ca-ATP) channel are administered.

Another aspect of the present invention comprises co-administration ofan antagonist of the NC_(Ca-ATP) channel with a thrombolytic agent.Co-administration of these two compound increases the therapeutic windowof the thrombolytic agent by reducing hemorrhagic conversion. Thetherapeutic window for thrombolytic agents may be increased by several(4-8) hours by co-administering antagonist of the NC_(Ca-ATP) channel.In addition to a thrombolytic agent, other agents can be used incombination with the antagonist of the present invention, for example,but not limited to antiplatelets, anticoagulants, vasodilators, statins,diuretics, etc.

Another aspect of the present invention comprises the use of labeledSUR1 antagonists to diagnose, determine or monitor stages of stroke,cerebral edema or visualize the size/boundaries/borders of a tumorand/or the stroke. For example, the penumbra following the stroke may bemonitored or visualized using labeled SUR1 antagonists.

Yet further, the compositions of the present invention can be used toproduce neuroprotective kits that are used to treat subjects at risk orsuffering from conditions that are associated with cytotoxic cerebraledema.

V. EXEMPLARY METHODS OF THE PRESENT INVENTION

The present invention provides a previously unknown ion channel found inmammalian neural cells that plays a role in cell swelling and brainswelling. The present invention further provides a method of screeningfor antagonists to the channel and a new use for antagonists to thechannel, including sulfonylurea compounds such as glibenclamide andtolbutamide, as a treatment for brain swelling in mammals.

Methods of the present invention for identifying compounds that interactwith, (e.g., bind to, open, block) the NC_(Ca-ATP) channel and employ(i) cell based assays and/or (ii) non-cell based assay systems. Suchcompounds may act as antagonists or agonists of NC_(Ca-ATP) channelactivity. In a preferred embodiment of the present invention,antagonists that block and/or inhibit the permeability of theNC_(Ca-ATP) channel are utilized in methods for treating neural cellswelling and/or brain swelling.

The cell based assays use neural cells that express the NC_(Ca-ATP)channel, preferably a functional NC_(Ca-ATP) channel; the preferredcells are NRAs. The non-cell based assay systems include membranepreparations that express the NC_(Ca-ATP) channel, preferably afunctional NC_(Ca-ATP) channel. Cell-based assays include, but are notlimited to, compound binding assays, microscopic observation of cellstatus (normal, blebbing, swelling, cell death), and measuring channelcurrents both before and after exposure to compound. Compositionscomprising membrane preparations expressing the NC_(Ca-ATP) channel maybe used to identify compounds that interact with, bind to, block or openthe NC_(Ca-ATP) channel or SUR1. The term “expressing the NC_(Ca-ATP)channel” or “expresses the NC_(Ca-ATP) channel” means having afunctional NC_(Ca-ATP) channel. The term “functional NC_(Ca-ATP)channel” as used herein means an NC_(Ca-ATP) channel capable of beingdetected. One preferred method of detecting the NC_(Ca-ATP) channel isby determining, in vitro or in vivo, whether the channel is open, closedand/or blocked.

For example, in a typical experiment using a membrane preparation, NRAsthat express the NC_(Ca-ATP) channel are used to produce the membranepreparation. Methods for producing membranes from whole cells andtissues are well known in the art. One such method produces purifiedcell membranes in the form of a purified microsomal fraction isolatedfrom disrupted cells or a tissue sample by discontinuous sucrosegradient centrifugation. Also included are membranes comprised ofcell-attached patches, inside-out patches, or outside-out patches. Oneexample of a tissue sample expressing NC_(Ca-ATP) channels is braintissue adjacent to brain injury.

The membrane preparations are used in a number of assays, including, butnot limited to measuring channel currents, both before and afterexposure to compound; and in vitro binding assays. The experimentalconditions for such assays to determine and quantify the status of theNC_(Ca-ATP) channel are described throughout the instant specification,including binding assay conditions, bath compositions, pipettesolutions, concentrations of ATP and Ca²⁺ required, membrane voltage,membrane potentials, compound quantity ranges, controls, etc.

Binding assays and competitive binding assays employ a labeled ligand orantagonist of the NC_(Ca-ATP) channel. In one such experiment, labeledGlibenclamide, such as FITC-conjugated glibenclamide orBODIPY-conjugated glibenclamide or radioactively labeled glibenclamideis bound to the membranes and assayed for specific activity; specificbinding is determined by comparison with binding assays performed in thepresence of excess unlabelled antagonist.

In one method for identifying NC_(Ca-ATP) channel blockers, membranesare incubated with a labeled compound shown to block this channel, ineither the presence or absence of test compound. Compounds that blockthe NC_(Ca-ATP) channel and compete with the labeled compound forbinding to the membranes will have a reduced signal, as compared to thevehicle control samples. In another aspect of the invention the screensmay be designed to identify compounds that compete with the interactionbetween NC_(Ca-ATP) channel and a known (previously identified herein)NC_(Ca-ATP) channel antagonist or SUR1 antagonist, such asglibenclamide. In such screens, the known NC_(Ca-ATP) channel antagonistor SUR1 antagonist is labeled and the test compounds are then assayedfor their ability to compete with or antagonize the binding of thelabeled antagonist.

The assays described herein can be used to identify compounds thatmodulate or affect NC_(Ca-ATP) channel activity. For example, compoundsthat affect NC_(Ca-ATP) channel activity include but are not limited tocompounds that bind to the NC_(Ca-ATP) channel or SUR1, inhibit bindingof identified blockers or ligands (such as glibenclamide), and eitheropen/activate the channel (agonists) or block/inhibit the channel(antagonists).

Assays described can also identify compounds that modulate neural cellswelling (e.g., compounds which affect other events involved in neuralcell swelling that are activated by ligand binding to or blocking of theNC_(Ca-ATP) channel).

VI. COMPOUNDS SCREENED IN ACCORDANCE WITH THE INVENTION

The compounds for screening in accordance with the invention include,but are not limited to organic compounds, peptides, antibodies andfragments thereof, peptidomimetics, that bind to the NC_(Ca-ATP) channeland either open the channel (i.e., agonists) or block the channel (i.e.,antagonists). For use in the treatment of neural cell swelling or brainswelling, compounds that block the channel are preferred. Agonists thatopen or maintain the channel in the open state include peptides,antibodies or fragments thereof, and other organic compounds thatinclude the SUR1 subunit of the NC_(Ca-ATP) channel (or a portionthereof) and bind to and “neutralize” circulating ligand for SUR1.

With reference to screening of compounds that affect the NC_(Ca-ATP)channel, libraries of known compounds can be screened, including naturalproducts or synthetic chemicals, and biologically active materials,including proteins, for compounds which are inhibitors or activators.Preferably, such a compound is an NC_(Ca-ATP) antagonist, which includesan NC_(Ca-ATP) channel inhibitor, an NC_(Ca-ATP) channel blocker, a SUR1antagonist, SUR1 inhibitor, and/or a compound capable of reducing themagnitude of membrane current through the channel.

Compounds may include, but are not limited to, small organic orinorganic molecules, compounds available in compound libraries, peptidessuch as, for example, soluble peptides, including but not limited tomembers of random peptide libraries; (see, e.g., Lam, K. S. et al.,1991, Nature 354: 82-84; Houghten, R. et al., 1991, Nature 354: 84-86),and combinatorial chemistry-derived molecular library made of D- and/orL-configuration amino acids, phosphopeptides (including, but not limitedto, members of random or partially degenerate, directed phosphopeptidelibraries; see, e.g., Songyang, Z. et al., 1993, Cell 72: 767-778),antibodies (including, but not limited to, polyclonal, monoclonal,humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab′).sub.2 and FAb expression library fragments, and epitope-bindingfragments thereof).

Other compounds which can be screened in accordance with the inventioninclude but are not limited to small organic molecules that may or maynot be able to cross the blood-brain barrier, gain entry into anappropriate neural cell and affect the expression of the NC_(Ca-ATP)channel gene or some other gene involved in the NC_(Ca-ATP) channelactivity (e.g., by interacting with the regulatory region ortranscription factors involved in gene expression); or such compoundsthat affect the activity of the NC_(Ca-ATP) channel or the activity ofsome other intracellular factor involved in the NC_(Ca-ATP) channelactivity.

Computer modeling and searching technologies permit identification ofcompounds, or the improvement of already identified compounds, that canmodulate NC_(Ca-ATP) channel activity or expression. Having identifiedsuch a compound or composition, the active sites or regions areidentified. Such active sites might typically be ligand binding sites.The active site can be identified using methods known in the artincluding, for example, from study of complexes of the relevant compoundor composition with other ligands, from the amino acid sequences ofpeptides, or from the nucleotide sequences of nucleic acids. Chemical orX-ray crystallographic methods can be used to study complexes of therelevant compound to find the active site. The three dimensionalgeometric structure of the active site is determined. This can be doneby known methods, including X-ray crystallography, which can determine acomplete molecular structure. On the other hand, solid or liquid phaseNMR can be used to determine certain intra-molecular distances. Anyother experimental method of structure determination can be used toobtain partial or complete geometric structures. The geometricstructures may be measured with a complexed ligand, natural which mayincrease the accuracy of the active site structure determined.

If an incomplete or insufficiently accurate structure is determined, themethods of computer based numerical modeling can be used to complete thestructure or improve its accuracy. Any recognized modeling method may beused, including parameterized models specific to particular biopolymerssuch as proteins or nucleic acids, molecular dynamics models based oncomputing molecular motions, statistical mechanics models based onthermal ensembles, or combined models. For most types of models,standard molecular force fields, representing the forces betweenconstituent atoms and groups, are necessary, and can be selected fromforce fields known in physical chemistry. The incomplete or lessaccurate experimental structures can serve as constraints on thecomplete and more accurate structures computed by these modelingmethods.

Finally, having determined the structure of the active site, eitherexperimentally, by modeling, or by a combination, candidate modulatingcompounds can be identified by searching databases containing compoundsalong with information on their molecular structure. Such a search seekscompounds having structures that match the determined active sitestructure and that interact with the groups defining the active site.Such a search can be manual, but is preferably computer assisted. Thesecompounds found from this search are potential NC_(Ca-ATP) channelmodulating, preferably blocking, compounds.

Alternatively, these methods can be used to identify improved modulatingcompounds from an already known modulating compound or ligand. Thecomposition of the known compound can be modified and the structuraleffects of modification can be determined using the experimental andcomputer modeling methods described above applied to the newcomposition. The altered structure is then compared to the active sitestructure of the compound to determine if an improved fit or interactionresults. In this manner systematic variations in composition, such as byvarying side groups, can be quickly evaluated to obtain modifiedmodulating compounds or ligands of improved specificity or activity.

Examples of molecular modeling systems are the CHARMm and QUANTAprograms (Polygen Corporation, Waltham, Mass.). CHARMm performs theenergy minimization and molecular dynamics functions. QUANTA performsthe construction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other. A number ofarticles review computer modeling of drugs interactive with specificproteins, such as Rotivinen, et al.) 1988, Acta Pharmaceutical Fennica97: 159-166); Ripka (1988 New Scientist 54-57); McKinaly and Rossmann(1989, Annu. Rev. Pharmacol. Toxicol. 29: 11 1-122); Perry and Davies,OSAR: Quantitative Structure-Activity Relationships in Drug Design pp.189-193 Alan R. Liss, Inc. 1989; Lewis and Dean (1989, Proc. R. SOC.Lond. 236: 125-140 and 141-162); and, with respect to a model receptorfor nucleic acid components, Askew, et al. (1989, J. Am. Chem. SOC. 111: 1082-1090). Other computer programs that screen and graphicallydepict chemicals are available from companies such as BioDesign, Inc.(Pasadena, Calif.), Allelix, Inc. (Mississauga, Ontario, Canada), andHypercube, Inc. (Cambridge, Ontario). Although these are primarilydesigned for application to drugs specific to particular proteins, theycan be adapted to design of drugs specific to regions of DNA or RNA,once that region is identified.

Compounds identified via assays such as those described herein may beuseful, for example, in elaborating the biological function of theNC_(Ca-ATP) channel and for relief of brain swelling.

Assays for testing the efficacy of compounds identified in the cellularscreen can be tested in animal model systems for brain or spinal cordswelling. Such animal models may be used as test substrates for theidentification of drugs, pharmaceuticals, therapies and interventionswhich may be effective in treating brain or spinal cord swelling. Forexample, animal models of brain swelling, such as brain injury, may beexposed to a compound, suspected of exhibiting an ability to inhibitbrain swelling, at a sufficient concentration and for a time sufficientto elicit such an inhibition of brain swelling in the exposed animals.The response of the animals to the exposure may be monitored usingvisual means (e.g., radiological, CAT, MRI), measurement of intracranialpressure, and/or the reversal of symptoms associated with brainswelling. With regard to intervention, any treatments which reverse anyaspect of brain swelling-associated symptoms should be considered ascandidates for brain swelling therapeutic intervention. Dosages of testagents may be determined by deriving dose-response curves, as discussedherein.

Accordingly, the present invention is useful in the treatment oralleviation of neural cell swelling and death and brain swelling,especially those brain insults related to traumatic brain injury, spinalcord injury, central or peripheral nervous system damage, cerebralischemia, such as stroke, or complications involving and/or stemmingfrom edema, injury, or trauma. Such damage or complications may becharacterized by an apparent brain damage or aberration, the symptoms ofwhich can be reduced by the methods of the present invention includingthe administration of an effective amount of the active compounds orsubstances described herein. According to a specific embodiment of thepresent invention the administration of effective amounts of the activecompound can block the channel, which if it remained open would lead toneural cell swelling and cell death. A variety of antagonists to SUR1are suitable for blocking the channel. Examples of suitable SUR1antagonists include, but are not limited to glibenclamide, tolbutamide,repaglinide, nateglinide, meglitinide, midaglizole, LY397364, LY3 89382,gliclazide, glimepiride, MgADP, and combinations thereof. In a preferredembodiment of the invention the SUR1 antagonists is selected from thegroup consisting of glibenclamide and tolbutamide. Still othertherapeutic “strategies” for preventing neural cell swelling and celldeath can be adopted including, but not limited to methods that maintainthe neural cell in a polarized state and methods that prevent strongdepolarization.

A. Modulators of the NC_(Ca-ATP) Channel

The present invention comprises modulators of the channel, for exampleone or more agonists and/or one or more antagonists of the channel.Examples of antagonists or agonists of the present invention mayencompass respective antagonists and/or agonists identified in USApplication Publication No. 20030215889, which is incorporated herein byreference in its entirety. One of skill in the art is aware that theNC_(Ca-ATP) channel is comprised of at least two subunits: theregulatory subunit, SUR1, and the pore forming subunit.

B. Modulators of SUR1

In certain embodiments, antagonists to sulfonylurea receptor-1 (SUR1)are suitable for blocking the channel. Examples of suitable SUR1antagonists include, but are not limited to glibenclamide, tolbutamide,repaglinide, nateglinide, meglitinide, midaglizole, LY397364, LY389382,glyclazide, glimepiride, estrogen, estrogen related-compounds estrogenrelated-compounds (estradiol, estrone, estriol, genistein, non-steroidalestrogen (e.g., diethystilbestrol), phytoestrogen (e.g., coumestrol),zearalenone, etc.) and combinations thereof. In a preferred embodimentof the invention the SUR1 antagonists is selected from the groupconsisting of glibenclamide and tolbutamide. Yet further, anotherantagonist can be MgADP. Other antagonist include blockers of K_(ATP)channels, for example, but not limited to tolbutamide, glyburide(1[p-2[5-chloro-O-anisamido)ethyl]phenyl]sulfonyl]-3-cyclohexyl-3-urea);chlorpropamide (1-[[(p-chlorophenyl)sulfonyl]-3-propylurea; glipizide(1-cyclohexyl-3[[p-[2(5-methylpyrazinecarboxamido)ethyl]phenyl]sulfonyl]urea); ortolazamide(benzenesulfonamide-N-[[(hexahydro-1H-azepin-1yl)amino]carbonyl]-4-methyl).

Agonists that may be used in the present invention include, but are notlimited to, one or more agonists of SUR1, for example, diazoxide,pinacidil, P1075, cromakalin or activators of K_(ATP) channels. Otheragonists can include, but are not limited to diazoxide derivatives, forexample 3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide(NNC 55-9216), 6,7-dichloro-3-isopropylamino-4H-1,2,4-benzothiadiazine1,1-dioxide (BPDZ 154),7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (BPDZ73), 6-Chloro-3-isopropylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine1,1-dioxide (NNC 55-0118)4,6-chloro-3-(1-methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine1,1-dioxide (NN414),3-(3-methyl-2-butylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide(BPDZ 44),3-(1′,2′,2′-trimethylpropyl)amino-4H-pyrido(4,3-e)-1,2,4-thiadiazine1,1-dioxide (BPDZ 62), 3-(1′,2′,2′-trimethylpropyl)amine-4H-pyrido(2,3-e)-1,2,4-thiadiazine, 1,1-dioxide (BPDZ 79),2-alkyl-3-alkylamino-2H-benzo- and2-alkyl-3-alkylamino-2H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides,6-Chloro-3-alkylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxidederivatives, 4-N-Substituted and -unsubstituted 3-alkyl- and3-(alkylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides. Inaddition, other compounds, including 6-chloro-2-methylquinolin-4(1H)-one(HEI 713) and LN 533021, as well as the class of drugs,arylcyanoguanidines, are known activators or agonist of SUR1.

C. Modulators of SUR1 Transcription and/or Translation

In certain embodiments, the modulator can comprise a compound (protein,nucleic acid, siRNA, etc.) that modulates transcription and/ortranslation of SUR1 (regulatory subunit) and/or the molecular entitiesthat comprise the pore-forming subunit.

D. Transcription Factors

Transcription factors are regulatory proteins that binds to a specificDNA sequence (e.g., promoters and enhancers) and regulate transcriptionof an encoding DNA region. Thus, transcription factors can be used tomodulate the expression of SUR1. Typically, a transcription factorcomprises a binding domain that binds to DNA (a DNA-binding domain) anda regulatory domain that controls transcription. Where a regulatorydomain activates transcription, that regulatory domain is designated anactivation domain. Where that regulatory domain inhibits transcription,that regulatory domain is designated a repression domain. Morespecifically, transcription factors such as Sp1, HIF1α, and NFκB can beused to modulate expression of SUR1.

In particular embodiments of the invention, a transcription factor maybe targeted by a composition of the invention. The transcription factormay be one that is associated with a pathway in which SUR1 is involved.The transcription factor may be targeted with an antagonist of theinvention, including siRNA to downregulate the transcription factor.Such antagonists can be identified by standard methods in the art, andin particular embodiments the antagonist is employed for treatment andor prevention of an individual in need thereof. In an additionalembodiment, the antagonist is employed in conjunction with an additionalcompound, such as a composition that modulates the NC_(CA-ATP) channelof the invention. For example, the antagonist may be used in combinationwith an inhibitor of the channel of the invention. When employed incombination, the antagonist of a transcription factor of a SUR1-relatedpathway may be administered prior to, during, and/or subsequent to theadditional compound.

E. Antisense and Ribozymes

An antisense molecule that binds to a translational or transcriptionalstart site, or splice junctions, are ideal inhibitors. Antisense,ribozyme, and double-stranded RNA molecules target a particular sequenceto achieve a reduction or elimination of a particular polypeptide, suchas SUR1. Thus, it is contemplated that antisense, ribozyme, anddouble-stranded RNA, and RNA interference molecules are constructed andused to modulate SUR1 expression.

F. Antisense Molecules

Antisense methodology takes advantage of the fact that nucleic acidstend to pair with complementary sequences. By complementary, it is meantthat polynucleotides are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,the larger purines will base pair with the smaller pyrimidines to formcombinations of guanine paired with cytosine (G:C) and adenine pairedwith either thymine (A:T) in the case of DNA, or adenine paired withuracil (A:U) in the case of RNA. Inclusion of less common bases such asinosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others inhybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNAs, are employed to inhibit gene transcription or translation or bothwithin a host cell, either in vitro or in vivo, such as within a hostanimal, including a human subject.

Antisense constructs are designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. It is contemplated that the most effective antisense constructsmay include regions complementary to intron/exon splice junctions. Thus,antisense constructs with complementarity to regions within 50-200 basesof an intron-exon splice junction are used. It has been observed thatsome exon sequences can be included in the construct without seriouslyaffecting the target selectivity thereof. The amount of exonic materialincluded will vary depending on the particular exon and intron sequencesused. One can readily test whether too much exon DNA is included simplyby testing the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

It is advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to generate specific constructs. For example, wherean intron is desired in the ultimate construct, a genomic clone willneed to be used. The cDNA or a synthesized polynucleotide may providemore convenient restriction sites for the remaining portion of theconstruct and, therefore, would be used for the rest of the sequence.

G. RNA Interference

It is also contemplated in the present invention that double-strandedRNA is used as an interference molecule, e.g., RNA interference (RNAi).RNA interference is used to “knock down” or inhibit a particular gene ofinterest by simply injecting, bathing or feeding to the organism ofinterest the double-stranded RNA molecule. This technique selectively“knock downs” gene function without requiring transfection orrecombinant techniques (Giet, 2001; Hammond, 2001; Stein P, et al.,2002; Svoboda P, et al., 2001; Svoboda P, et al., 2000).

Another type of RNAi is often referred to as small interfering RNA(siRNA), which may also be utilized to inhibit SUR1. A siRNA maycomprises a double stranded structure or a single stranded structure,the sequence of which is “substantially identical” to at least a portionof the target gene (See WO 04/046320, which is incorporated herein byreference in its entirety). “Identity,” as known in the art, is therelationship between two or more polynucleotide (or polypeptide)sequences, as determined by comparing the sequences. In the art,identity also means the degree of sequence relatedness betweenpolynucleotide sequences, as determined by the match of the order ofnucleotides between such sequences. Identity can be readily calculated.See, for example: Computational Molecular Biology, Lesk, A.M., ed.Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ea., Academic Press, New York, 1993, andthe methods disclosed in WO 99/32619, WO 01/68836, WO 00/44914, and WO01/36646, specifically incorporated herein by reference. While a numberof methods exist for measuring identity between two nucleotidesequences, the term is well known in the art. Methods for determiningidentity are typically designed to produce the greatest degree ofmatching of nucleotide sequence and are also typically embodied incomputer programs. Such programs are readily available to those in therelevant art. For example, the GCG program package (Devereux et al.),BLASTP, BLASTN, and FASTA (Atschul et al.,) and CLUSTAL (Higgins et al.,1992; Thompson, et al., 1994).

Thus, siRNA contains a nucleotide sequence that is essentially identicalto at least a portion of the target gene, for example, SUR1, or anyother molecular entity associated with the NC_(Ca-ATP) channel such asthe pore-forming subunit. One of skill in the art is aware that thenucleic acid sequences for SUR1 are readily available in GenBank, forexample, GenBank accession L40624, which is incorporated herein byreference in its entirety. Preferably, the siRNA contains a nucleotidesequence that is completely identical to at least a portion of thetarget gene. Of course, when comparing an RNA sequence to a DNAsequence, an “identical” RNA sequence will contain ribonucleotides wherethe DNA sequence contains deoxyribonucleotides, and further that the RNAsequence will typically contain a uracil at positions where the DNAsequence contains thymidine.

One of skill in the art will appreciate that two polynucleotides ofdifferent lengths may be compared over the entire length of the longerfragment. Alternatively, small regions may be compared. Normallysequences of the same length are compared for a final estimation oftheir utility in the practice of the present invention. It is preferredthat there be 100% sequence identity between the dsRNA for use as siRNAand at least 15 contiguous nucleotides of the target gene (e.g., SUR1),although a dsRNA having 70%, 75%, 80%, 85%, 90%, or 95% or greater mayalso be used in the present invention. A siRNA that is essentiallyidentical to a least a portion of the target gene may also be a dsRNAwherein one of the two complementary strands (or, in the case of aself-complementary RNA, one of the two self-complementary portions) iseither identical to the sequence of that portion or the target gene orcontains one or more insertions, deletions or single point mutationsrelative to the nucleotide sequence of that portion of the target gene.siRNA technology thus has the property of being able to toleratesequence variations that might be expected to result from geneticmutation, strain polymorphism, or evolutionary divergence.

There are several methods for preparing siRNA, such as chemicalsynthesis, in vitro transcription, siRNA expression vectors, and PCRexpression cassettes. Irrespective of which method one uses, the firststep in designing an siRNA molecule is to choose the siRNA target site,which can be any site in the target gene. In certain embodiments, one ofskill in the art may manually select the target selecting region of thegene, which may be an ORF (open reading frame) as the target selectingregion and may preferably be 50-100 nucleotides downstream of the “ATG”start codon. However, there are several readily available programsavailable to assist with the design of siRNA molecules, for examplesiRNA Target Designer by Promega, siRNA Target Finder by GenScriptCorp., siRNA Retriever Program by Imgenex Corp., EMBOSS siRNA algorithm,siRNA program by Qiagen, Ambion siRNA predictor, Ambion siRNA predictor,Whitehead siRNA prediction, and Sfold. Thus, it is envisioned that anyof the above programs may be utilized to produce siRNA molecules thatcan be used in the present invention.

H. Ribozymes

Ribozymes are RNA-protein complexes that cleave nucleic acids in asite-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim and Cech, 1987; Forster and Symons,1987). For example, a large number of ribozymes accelerate phosphoestertransfer reactions with a high degree of specificity, often cleavingonly one of several phosphoesters in an oligonucleotide substrate (Cechet al., 1981; Reinhold-Hurek and Shub, 1992). This specificity has beenattributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part of sequencespecific cleavage/ligation reactions involving nucleic acids (Joyce,1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855 reportsthat certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of gene expression is particularly suitedto therapeutic applications (Scanlon et al., 1991; Sarver et al., 1990;Sioud et al., 1992). Most of this work involved the modification of atarget mRNA, based on a specific mutant codon that is cleaved by aspecific ribozyme. In light of the information included herein and theknowledge of one of ordinary skill in the art, the preparation and useof additional ribozymes that are specifically targeted to a given genewill now be straightforward.

Other suitable ribozymes include sequences from RNase P with RNAcleavage activity (Yuan et al., 1992; Yuan and Altman, 1994), hairpinribozyme structures (Berzal-Herranz et al., 1992; Chowrira et al., 1993)and hepatitis 8 virus based ribozymes (Perrotta and Been, 1992). Thegeneral design and optimization of ribozyme directed RNA cleavageactivity has been discussed in detail (Haseloff and Gerlach, 1988;Symons, 1992; Chowrira, et al., 1994; and Thompson, et al., 1995).

The other variable on ribozyme design is the selection of a cleavagesite on a given target RNA. Ribozymes are targeted to a given sequenceby virtue of annealing to a site by complimentary base pairinteractions. Two stretches of homology are required for this targeting.These stretches of homologous sequences flank the catalytic ribozymestructure defined above. Each stretch of homologous sequence can vary inlength from 7 to 15 nucleotides. The only requirement for defining thehomologous sequences is that, on the target RNA, they are separated by aspecific sequence which is the cleavage site. For hammerhead ribozymes,the cleavage site is a dinucleotide sequence on the target RNA, uracil(U) followed by either an adenine, cytosine or uracil (A, C or U;Perriman, et al., 1992; Thompson, et al., 1995). The frequency of thisdinucleotide occurring in any given RNA is statistically 3 out of 16.

Designing and testing ribozymes for efficient cleavage of a target RNAis a process well known to those skilled in the art. Examples ofscientific methods for designing and testing ribozymes are described byChowrira et al. (1994) and Lieber and Strauss (1995), each incorporatedby reference. The identification of operative and preferred sequencesfor use in SUR1 targeted ribozymes is simply a matter of preparing andtesting a given sequence, and is a routinely practiced screening methodknown to those of skill in the art.

I. Inhibition of Post-Translational Assembly and Trafficking

Following expression of individual regulatory and pore-forming subunitproteins of the channel, and in particular aspects of the invention,these proteins are modified by glycosylation in the Golgi apparatus ofthe cell, assembled into functional heteromultimers that comprise thechannel, and then transported to the plasmalemmal membrane where theyare inserted to form functional channels. The last of these processes isreferred to as “trafficking”.

In specific embodiments of the invention, molecules that bind to any ofthe constituent proteins interfere with post-translational assembly andtrafficking, and thereby interfere with expression of functionalchannels. One such example is with glibenclamide binding to SUR1subunits. In additional embodiments, glibenclamide, which binds withfemtomolar affinity to SUR1, interferes with post-translational assemblyand trafficking required for functional channel expression.

VII. EXEMPLARY METHODS OF SCREENING FOR MODULATORS

Further embodiments of the present invention can include methods foridentifying modulators of the NC_(Ca-ATP) channel, for example, agonistor antagonist, that modify the activity and/or expression. These assaysmay comprise random screening of large libraries of candidatesubstances; alternatively, the assays may be used to focus on particularclasses of compounds selected with an eye towards structural attributesthat are believed to make them more likely to modulate the function oractivity or expression of the NC_(Ca-ATP) channel.

By function, it is meant that one may assay for mRNA expression, proteinexpression, protein activity, or channel activity, more specifically,the ability of the modulator to open or inhibit or block the NC_(Ca-ATP)channel. Thus, the compounds for screening in accordance with theinvention include, but are not limited to natural or synthetic organiccompounds, peptides, antibodies and fragments thereof, peptidomimetics,that bind to the NC_(Ca-ATP) channel and either open the channel (e.g.,agonists) or block the channel (e.g., antagonists). For use in thetreatment of neural cell swelling or brain swelling, compounds thatblock the channel are preferred. Agonists that open or maintain thechannel in the open state include peptides, antibodies or fragmentsthereof, and other organic compounds that include the SUR1 subunit ofthe NC_(Ca-ATP) channel (or a portion thereof) and bind to and“neutralize” circulating ligand for SUR1.

With reference to screening of compounds that affect the NC_(Ca-ATP)channel, libraries of known compounds can be screened, including naturalproducts or synthetic chemicals, and biologically active materials,including proteins, for compounds which are inhibitors or activators.Preferably, such a compound is an NC_(Ca-ATP) antagonist, which includesan NC_(Ca-ATP) channel inhibitor, an NC_(Ca-ATP) channel blocker, a SUR1antagonist, SUR1 inhibitor, and/or a compound capable of reducing themagnitude of membrane current through the channel.

Compounds may include, but are not limited to, small organic orinorganic molecules, compounds available in compound libraries, peptidessuch as, for example, soluble peptides, including but not limited tomembers of random peptide libraries; (see, e.g., Lam, K. S. et al.,1991, Nature 354: 82-84; Houghten, R. et al., 1991, Nature 354: 84-86),and combinatorial chemistry-derived molecular library made of D- and/orL-configuration amino acids, phosphopeptides (including, but not limitedto, members of random or partially degenerate, directed phosphopeptidelibraries; see, e.g., Songyang, Z. et al., 1993, Cell 72: 767-778),antibodies (including, but not limited to, polyclonal, monoclonal,humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab′)₂ and FAb expression library fragments, and epitope-bindingfragments thereof).

Other compounds that can be screened in accordance with the inventioninclude but are not limited to small organic molecules that may or maynot cross the blood-brain barrier, gain entry into an appropriate neuralor endothelial cell and affect the expression of the NC_(Ca-ATP) channelgene or some other gene involved in the NC_(Ca-ATP) channel activity(e.g., by interacting with the regulatory region or transcriptionfactors involved in gene expression, or by interfering withpost-translational channel assembly or trafficking); or such compoundsthat affect the activity of the NC_(Ca-ATP) channel or the activity ofsome other intracellular factor involved in the NC_(Ca-ATP) channelactivity.

To identify, make, generate, provide, manufacture or obtain modulator,one generally will determine the activity of the NC_(Ca-ATP) channel inthe presence, absence, or both of the candidate substance, wherein aninhibitor or antagonist is defined as any substance that down-regulates,reduces, inhibits, blocks or decreases the NC_(Ca-ATP) channelexpression or activity, and wherein an activator or agonist is definedas any substance that up-regulates, enhances, activates, increases oropens the NC_(Ca-ATP) channel. For example, a method may generallycomprise:

(a) providing a candidate substance suspected of activating orinhibiting the NC_(Ca-ATP) channel expression or activity in vitro or invivo;

(b) assessing the ability of the candidate substance to activate orinhibit the NC_(Ca-ATP) channel expression or activity in vitro or invivo;

(c) selecting a modulator; and

(d) manufacturing the modulator.

In certain embodiments, an alternative assessing step can be assessingthe ability of the candidate substance to bind specifically to theNC_(Ca-ATP) channel in vitro or in vivo;

In further embodiments, the NC_(Ca-ATP) channel may be provided in acell or a cell free system and the NC_(Ca-ATP) channel may be contactedwith the candidate substance. Next, the modulator is selected byassessing the effect of the candidate substance on the NC_(Ca-ATP)channel activity or expression. Upon identification of the modulator,the method may further provide manufacturing of the modulator.

An effective amount of modulator of an NC_(Ca-ATP) channel (which may bean agonist or antagonist, and is preferably an antagonist) that may beadministered to a cell includes a dose of about 0.0001 nM to about 2000μM. More specifically, doses of an agonist to be administered are fromabout 0.01 nM to about 2000 μM; about 0.01 μM to about 0.05 μM; about0.05 μM to about 1.0 μM; about 1.0 μM to about 1.5 μM; about 1.5 μM toabout 2.0 μM; about 2.0 μM to about 3.0 μM; about 3.0 μM to about 4.0μM; about 4.0 μM to about 5.0 μM; about 5.0 μM to about 10 μM; about 10μM to about 50 μM; about 50 μM to about 100 μM; about 100 μM to about200 μM; about 200 μM to about 300 μM; about 300 μM to about 500 μM;about 500 μM to about 1000 μM; about 1000 μM to about 1500 μM and about1500 μM to about 2000 μM. Of course, all of these amounts are exemplary,and any amount in-between these points is also expected to be of use inthe invention.

The NC_(Ca-ATP) channel modulator or related-compound thereof can beadministered parenterally or alimentarily. Parenteral administrationsinclude, but are not limited to intravenously, intradermally,intramuscularly, intraarterially, intrathecally, intraventricularly,intratumorally, subcutaneous, or intraperitoneally U.S. Pat. Nos.6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (eachspecifically incorporated herein by reference in its entirety).Alimentary administrations include, but are not limited to orally,buccally, rectally, or sublingually.

The administration of the therapeutic compounds and/or the therapies ofthe present invention may include systemic, local and/or regional andmay oral, intravenous, and intramuscular. Alternatively, other routes ofadministration are also contemplated such as, for example, arterialperfusion, intracavitary, intraperitoneal, intrapleural,intraventricular, intratumoral, intraparenchyma and/or intrathecal. Ifdesired the therapeutic compound may be administered by the same routeas the chemotherapeutic agent, even if the therapeutic compound and thechemotherapeutic agent are not administered simultaneously. The skilledartisan is aware of determining the appropriate administration routeusing standard methods and procedures. In one example, where assessmentof a response to chemotherapy, both peripherally and centrally isdesired, the health care professional may use a systemic administration.

Treatment methods will involve treating an individual with an effectiveamount of a composition containing an agonist of NC_(Ca-ATP) channel orrelated-compound thereof. An effective amount is described, generally,as that amount sufficient to detectably and repeatedly to ameliorate,reduce, minimize or limit the extent of a disease or its symptoms. Morespecifically, it is envisioned that the treatment with the an antagonistof NC_(Ca-ATP) channel or related-compounds thereof will reduce cellswelling and brain swelling following stroke, brain trauma, or otherbrain injury, and will reduce brain damage following stroke, braintrauma or other brain injury or spinal cord injury.

The effective amount of “therapeutically effective amounts” of the anantagonist of NC_(Ca-ATP) channel or related-compounds thereof to beused are those amounts effective to produce beneficial results,particularly with respect to stroke or brain trauma treatment, in therecipient animal or patient. Such amounts may be initially determined byreviewing the published literature, by conducting in vitro tests or byconducting metabolic studies in healthy experimental animals. Before usein a clinical setting, it may be beneficial to conduct confirmatorystudies in an animal model, preferably a widely accepted animal model ofthe particular disease to be treated. Preferred animal models for use incertain embodiments are rodent models, which are preferred because theyare economical to use and, particularly, because the results gained arewidely accepted as predictive of clinical value.

As is well known in the art, a specific dose level of active compoundssuch as an antagonist of NC_(Ca-ATP) channel or related-compoundsthereof for any particular patient depends upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination, and the severity ofthe particular disease undergoing therapy. The person responsible foradministration will determine the appropriate dose for the individualsubject. Moreover, for human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards.

An effective amount of an antagonist of NC_(Ca-ATP) channel orrelated-compounds thereof as a treatment varies depending upon the hosttreated and the particular mode of administration. In one embodiment ofthe invention the dose range of the agonist of NC_(Ca-ATP) channel orrelated-compounds thereof will be about 0.0001 μg/kg body weight toabout 500 mg/kg body weight. The term “body weight” is applicable whenan animal is being treated. When isolated cells are being treated, “bodyweight” as used herein should read to mean “total cell body weight”. Theterm “total body weight” may be used to apply to both isolated cell andanimal treatment. All concentrations and treatment levels are expressedas “body weight” or simply “kg” in this application are also consideredto cover the analogous “total cell body weight” and “total body weight”concentrations. However, those of skill will recognize the utility of avariety of dosage range, for example, 0.0001 μg/kg body weight to 450mg/kg body weight, 0.0002 μg/kg body weight to 400 mg/kg body weight,0.0003 μg/kg body weight to 350 mg/kg body weight, 0.0004 μg/kg bodyweight to 300 mg/kg body weight, 0.0005 μg/kg body weight to 250 mg/kgbody weight, 5.0 μg/kg body weight to 200 mg/kg body weight, 10.0 μg/kgbody weight to 150 mg/kg body weight, 100.0 μg/kg body weight to 100mg/kg body weight, or 1000 μg/kg body weight to 50 mg/kg body weight.Further, those of skill will recognize that a variety of differentdosage levels will be of use, for example, up to about 0.0001 μg/kg, upto about 0.0002 μg/kg, up to about 0.0003 μg/kg, less than about 0.0004μg/kg, less than about 0.005 μg/kg, less than about 0.0007 μg/kg, lessthan about 0.001 μg/kg, less than about 0.1 μg/kg, less than about 1.0μg/kg, less than about 1.5 μg/kg, less than about 2.0 μg/kg, less thanabout 5.0 μg/kg, less than about 10.0 μg/kg, less than about 15.0 μg/kg,less than about 30.0 μg/kg, less than about 50 μg/kg, less than about 75μg/kg, less than about 80 μg/kg, less than about 90 μg/kg, less thanabout 100 μg/kg, less than about 200 μg/kg, less than about 300 μg/kg,less than about 400 μg/kg, less than about 500 μg/kg, less than about 1mg/kg, less than about 2 mg/kg, less than about 3 mg/kg, less than about5 mg/kg, less than about 10 mg/kg, less than about 100 mg/kg. Further,those of skill will recognize that a variety of different dosage levelswill be of use, for example, 0.0001 μg/kg, 0.0002 μg/kg, 0.0003 μg/kg,0.0004 μg/kg, 0.005 μg/kg, 0.0007 μg/kg, 0.001 μg/kg, 0.1 μg/kg, 1.0μg/kg, 1.5 μg/kg, 2.0 μg/kg, 5.0 μg/kg, 10.0 μg/kg, 15.0 μg/kg, 30.0μg/kg, 50 μg/kg, 75 μg/kg, 80 μg/kg, 90 μg/kg, 100 μg/kg, 120 μg/kg, 140μg/kg, 150 μg/kg, 160 μg/kg, 180 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg,275 μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 450μg/kg, 500 μg/kg, 550 μg/kg, 600 μg/kg, 700 μg/kg, 750 μg/kg, 800 μg/kg,900 μg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 12 mg/kg, 15 mg/kg, 20 mg/kg,and/or 30 mg/kg. Of course, all of these dosages are exemplary, and anydosage in-between these points is also expected to be of use in theinvention. Any of the above dosage ranges or dosage levels may beemployed for an agonist of NC_(Ca-ATP) channel or related-compoundsthereof.

Administration of the therapeutic agonist of NC_(Ca-ATP) channelcomposition of the present invention to a patient or subject will followgeneral protocols for the administration of chemotherapeutics, takinginto account the toxicity, if any, of the agonist of NC_(Ca-ATP)channel. It is expected that the treatment cycles would be repeated asnecessary. It also is contemplated that various standard therapies, aswell as surgical intervention, may be applied in combination with thedescribed therapy.

The treatments may include various “unit doses.” Unit dose is defined ascontaining a predetermined quantity of the therapeutic composition (anagonist of NC_(Ca-ATP) channel or its related-compounds thereof)calculated to produce the desired responses in association with itsadministration, e.g., the appropriate route and treatment regimen. Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. Also of import isthe subject to be treated, in particular, the state of the subject andthe protection desired. A unit dose need not be administered as a singleinjection but may comprise continuous infusion over a set period oftime.

According to the present invention, one may treat stroke, brain trauma,or other brain or spinal cord injury by systemic administration, such asintravenous, intra-arterial, peritoneal, by administration via pump, orby direct injection into the brain or ventricles with an antagonist ofNC_(Ca-ATP) channel or related-compound composition. Alternatively, thebrain or spinal cord may be infused or perfused with the compositionusing any suitable delivery vehicle. Systemic administration or oraladministration may be performed, and, in embodiments of the presentinvention, local or regional administration may be performed. Continuousadministration also may be applied where appropriate, for example, wherea patient may be monitored on an on-going basis. Delivery via syringe orcatheterization is one effective method. Continuous perfusion may takeplace for a period from about 1-2 hours, to about 2-6 hours, to about6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1-2 wk orlonger following the initiation of treatment. Generally, the dose of thetherapeutic composition via continuous perfusion will be equivalent tothat given by a single or multiple injections, adjusted over a period oftime during which the perfusion occurs. Multiple injections delivered assingle dose comprise about 0.1 to about 1 ml volumes. In embodiments,the volume to be administered may be about 4-10 ml (preferably 10 ml),while in further embodiments a volume of about 1-3 ml will be used(preferably 3 ml).

VIII. METHODS OF CEREBRAL ISCHEMIA TREATMENT Treatment with anAntagonist

In other embodiments, the therapeutic compound of the present inventioncomprises an antagonist of a NC_(Ca-ATP) channel of a neuronal cell, aneuroglial cell, a neural endothelial cell or a combination thereof.Antagonists are contemplated for use in treating adverse conditionsassociated with intracranial pressure and/or ionic or cytotoxic edema ofthe central nervous system. Such conditions include trauma (e.g.,traumatic brain or spinal cord injury (TBI or SCI, respectively)),ischemic brain or spinal cord injury, primary and secondary neuronalinjury, stroke, arteriovenous malformations (AVM), mass-occupying lesion(e.g., hematoma), and hemorrhagic infarction. Antagonists protect thecells expressing the NC_(CA-ATP) channel, which is desirable forclinical treatment in which ionic or cytotoxic edema is formed, in whichcapillary integrity is lost following ischemia, and in which glioticcapsule integrity is important and must be maintained to prevent thespread of infection, such as with a brain abscess. Those of skill in theart realize that a brain abscess is a completely enclosed and results incerebral swelling. The protection via inhibition of the NC_(Ca-ATP)channel is associated with a reduction in cerebral ionic and cytotoxicedema. Thus, the compound that inhibits the NC_(Ca-ATP) channel isneuroprotective.

In one aspect, the NC_(Ca-ATP) channel is blocked, inhibited, orotherwise is decreased in activity. In such examples, an antagonist ofthe NC_(Ca-ATP) channel is administered and/or applied. The antagonistmodulates the NC_(Ca-ATP) channel such that flux (ion and/or water)through the channel is reduced, ceased, decreased and/or stopped. Theantagonist may have a reversible or an irreversible activity withrespect to the activity of the NC_(Ca-ATP) channel of the neuronal cell,neuroglial cell, a neural endothelial cell or a combination thereof.Thus, inhibition of the NC_(Ca-ATP) channel can reduce cytotoxic edemaand death of endothelial cells which are associated with formation ofionic edema and with hemorrhagic conversion.

Accordingly, the present invention is useful in the treatment oralleviation of acute cerebral ischemia. According to a specificembodiment of the present invention the administration of effectiveamounts of the active compound can block the channel, which if remainedopen leads to neuronal cell swelling and cell death. A variety ofantagonists to SUR1 are suitable for blocking the channel. Examples ofsuitable SUR1 antagonists include, but are not limited to glibenclamide,tolbutamide, repaglinide, nateglinide, meglitinide, midaglizole,LY397364, LY389382, glyclazide, glimepiride, estrogen, estrogenrelated-compounds and combinations thereof. In a preferred embodiment ofthe invention the SUR1 antagonists is selected from the group consistingof glibenclamide and tolbutamide. Another antagonist that can be used isMgADP. Still other therapeutic “strategies” for preventing neural cellswelling and cell death can be adopted including, but not limited tomethods that maintain the neural cell in a polarized state and methodsthat prevent strong depolarization.

In further embodiments, inhibitors or antagonist of the NC_(Ca-ATP)channel can be used to reduce or alleviate or abrogate hemorrhagicconversion. The pathological sequence that takes place in capillariesafter ischemia can be divided into 3 stages, based on the principalconstituents that move from the intravascular compartment into brainparenchyma (Ayata 2002; Betz, 1996; Betz 1989). The first stage ischaracterized by formation of “ionic” edema, during which the BBBremains intact, with movement of electrolytes (Na⁺, Cl⁻) plus water intobrain parenchyma. The second stage is characterized by formation of“vasogenic” edema, due to breakdown of the BBB, during whichmacromolecules plus water enter into brain parenchyma. The third stageis characterized by hemorrhagic conversion, due to catastrophic failureof capillaries, during which all constituents of blood extravasate intobrain parenchyma. In accordance with Starling's law, understanding thesephases requires that 2 things be identified: (i) the driving force that“pushes” things into parenchyma; and (ii) the permeability pore thatallows passage of these things into parenchyma.

Thus, the use of the antagonist or related-compounds thereof can reducethe mortality of a subject suffering from a stroke and/or rescue thepenumbra area or prevent damage in the penumbra area which comprisesareas of tissue that are at risk of becoming irreversibly damaged.

With the administration of an antagonist of the NC_(Ca-ATP) channel,endothelial cell depolarization is abrogated, slowed, reduced orinhibited due to the opening of the NC_(Ca-ATP) channel. Thus,abrogation of cell depolarization results in abrogation or inhibition ofNa⁺ influx, which prevents a change in osmotic gradient therebypreventing an influx of water into the endothelial cell and stoppingcell swelling, blebbing and cytotoxic edema. Thus, preventing orinhibiting or attenuating endothelial cell depolarization can prevent orreduce hemorrhagic conversion.

Neuronal cells in which the antagonist of the NC_(Ca-ATP) channel may beadministered may include any cell that expresses SUR1, for example anyneuronal cell, neuroglial cell or a neural endothelia cell.

Subjects that may be treated with the antagonist or related-compoundthereof include those that are suffering from or at risk of developingtrauma (e.g., traumatic brain or spinal cord injury (TBI or SCI)),ischemic brain or spinal cord injury, primary and secondary neuronalinjury, stroke, arteriovenous malformations (AVM), brain abscess,mass-occupying lesion, hemorrhagic infarction, or any other conditionassociated with cerebral hypoxia or cerebral ischemia resulting incerebral edema and/or increased intracranial pressure, for example, butnot limited to brain mass, brain edema, hematoma, end stage cerebraledema, encephalopathies, etc. Thus, the antagonist can be a therapeutictreatment in which the therapeutic treatment includes prophylaxis or aprophylactic treatment. The antagonist or related-compounds thereof areneuroprotective.

Other subjects that may be treated with the antagonist of the presentinvention include those subjects that are at risk or predisposed todeveloping a stroke. Such subjects can include, but are not limited tosubjects that suffer from atrial fibrillations, clotting disorders,and/or risk of pulmonary emboli.

In certain embodiments, a subject at risk for developing a stroke mayinclude subjects undergoing treatments, for example, but not limited tocerebral/endovascular treatments, surgery (e.g., craniotomy, cranialsurgery, removal of brain tumors (e.g., hematoma), coronary arterybypass grafting (CABG), angiography, stent replacement, other vascularsurgeries, and/or other CNS or neurological surgeries), and treatment ofmyocardial infarction (MI) with thrombolytics, as well as surgeries onaortic abdominal aneurysms and major vessels that provide blood supplyto the spinal cord. In such cases, the subject may be treated with theantagonist or related-compound of the present invention prior to theactual treatment. Pretreatment can include administration of theantagonist and/or related-compound months (1, 2, 3, etc.), weeks (1, 2,3, etc.), days (1, 2, 3, etc.), hours (1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12), or minutes (15, 30, 60, 90, etc.) prior to the actual treatmentor surgery. Treatment of the antagonist and/or related-compound cancontinue during the treatment and/or surgery and after the treatmentand/or surgery until the risk of developing a stroke in the subject isdecreased, lessened or alleviated.

In further embodiments, the antagonist of the present invention can begiven to a subject at risk of developing head/neck trauma, such as asubject involved in sports or other activities that have an increasedrisk of head/neck trauma.

An effective amount of an antagonist of the NC_(Ca-ATP) channel that maybe administered to a cell includes a dose of about 0.0001 nM to about2000 μM. More specifically, doses of an agonist to be administered arefrom about 0.01 nM to about 2000 μM; about 0.01 μM to about 0.05 μM;about 0.05 μM to about 1.0 μM; about 1.0 μM to about 1.5 μM; about 1.5μM to about 2.0 μM; about 2.0 μM to about 3.0 μM; about 3.0 μM to about4.0 μM; about 4.0 μM to about 5.0 μM; about 5.0 μM to about 10 μM; about10 μM to about 50 μM; about 50 μM to about 100 μM; about 100 μM to about200 μM; about 200 μM to about 300 μM; about 300 μM to about 500 μM;about 500 μM to about 1000 μM; about 1000 μM to about 1500 μM and about1500 μM to about 2000 μM. Of course, all of these amounts are exemplary,and any amount in-between these points is also expected to be of use inthe invention.

The antagonist or related-compound thereof can be administeredparenterally or alimentary. Parenteral administrations include, but arenot limited to intravenously, intradermally, intramuscularly,intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S.Pat. Nos. 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363(each specifically incorporated herein by reference in its entirety).Alimentary administrations include, but are not limited to orally,buccally, rectally, or sublingually.

The administration of the therapeutic compounds and/or the therapies ofthe present invention may include systemic, local and/or regionaladministrations, for example, topically (dermally, transdermally), viacatheters, implantable pumps, etc. Alternatively, other routes ofadministration are also contemplated such as, for example, arterialperfusion, intracavitary, intraperitoneal, intrapleural,intraventricular and/or intrathecal. The skilled artisan is aware ofdetermining the appropriate administration route using standard methodsand procedures. Other routes of administration are discussed elsewherein the specification and are incorporated herein by reference.

Treatment methods will involve treating an individual with an effectiveamount of a composition containing an antagonist of NC_(Ca-ATP) channelor related-compound thereof. An effective amount is described,generally, as that amount sufficient to detectably and repeatedly toameliorate, reduce, minimize or limit the extent of a disease or itssymptoms. More specifically, it is envisioned that the treatment withthe an antagonist of NC_(Ca-ATP) channel or related-compounds thereofwill inhibit cell depolarization, inhibit Na⁺ influx, inhibit an osmoticgradient change, inhibit water influx into the cell, inhibit cytotoxiccell edema, decrease stroke size, inhibit hemorrhagic conversion, anddecrease mortality of the subject.

The effective amount of an antagonist of NC_(Ca-ATP) channel orrelated-compounds thereof to be used are those amounts effective toproduce beneficial results, particularly with respect to stroketreatment, in the recipient animal or patient. Such amounts may beinitially determined by reviewing the published literature, byconducting in vitro tests or by conducting metabolic studies in healthyexperimental animals. Before use in a clinical setting, it may bebeneficial to conduct confirmatory studies in an animal model,preferably a widely accepted animal model of the particular disease tobe treated. Preferred animal models for use in certain embodiments arerodent models, which are preferred because they are economical to useand, particularly, because the results gained are widely accepted aspredictive of clinical value.

As is well known in the art, a specific dose level of active compoundssuch as an antagonist of the NC_(Ca-ATP) channel or related-compoundsthereof for any particular patient depends upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination, and the severity ofthe particular disease undergoing therapy. The person responsible foradministration will determine the appropriate dose for the individualsubject. Moreover, for human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards.

One of skill in the art realizes that the effective amount of theantagonist or related-compound thereof can be the amount that isrequired to achieve the desired result: reduction in the risk of stroke,reduction in intracranial pressure, reduction in cell death, reductionin stroke size, reduction in spinal cord injury, etc. This amount alsois an amount that maintains a reasonable level of blood glucose in thepatient, for example, the amount of the antagonist maintains a bloodglucose level of at least 60 mmol/l, more preferably, the blood glucoselevel is maintain in the range of about 60 mmol/l to about 150 mmol/l.Thus, the amounts prevents the subject from becoming hypoglycemic. Ifglucose levels are not normal, then one of skill in the art wouldadminister either insulin or glucose, depending upon if the patient ishypoglycemic or hyperglycemic.

Administration of the therapeutic antagonist of NC_(Ca-ATP) channelcomposition of the present invention to a patient or subject will followgeneral protocols for the administration of therapies used in stroketreatment, such as thrombolytics, taking into account the toxicity, ifany, of the antagonist of the NC_(Ca-ATP) channel. It is expected thatthe treatment cycles would be repeated as necessary. It also iscontemplated that various standard therapies, as well as surgicalintervention, may be applied in combination with the described therapy.

IX. PHARMACEUTICAL FORMULATIONS AND METHODS OF TREATING NEURAL CELLSWELLING AND BRAIN SWELLING A. Compositions of the Present Invention

The present invention also contemplates therapeutic methods employingcompositions comprising the active substances disclosed herein.Preferably, these compositions include pharmaceutical compositionscomprising a therapeutically effective amount of one or more of theactive compounds or substances along with a pharmaceutically acceptablecarrier.

As used herein, the term “pharmaceutically acceptable” carrier means anon-toxic, inert solid, semi-solid liquid filler, diluent, encapsulatingmaterial, formulation auxiliary of any type, or simply a sterile aqueousmedium, such as saline. Some examples of the materials that can serve aspharmaceutically acceptable carriers are sugars, such as lactose,glucose and sucrose, starches such as corn starch and potato starch,cellulose and its derivatives such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt,gelatin, talc; excipients such as cocoa butter and suppository waxes;oils such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol,polyols such as glycerin, sorbitol, mannitol and polyethylene glycol;esters such as ethyl oleate and ethyl laurate, agar; buffering agentssuch as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcoholand phosphate buffer solutions, as well as other non-toxic compatiblesubstances used in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfateand magnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. Examples ofpharmaceutically acceptable antioxidants include, but are not limitedto, water soluble antioxidants such as ascorbic acid, cysteinehydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite,and the like; oil soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, aloha-tocopherol and the like; and the metalchelating agents such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

B. Dose Determinations

By a “therapeutically effective amount” or simply “effective amount” ofan active compound, such as glibenclamide or tolbutamide, is meant asufficient amount of the compound to treat or alleviate the brainswelling at a reasonable benefit/risk ratio applicable to any medicaltreatment. It will be understood, however, that the total daily usage ofthe active compounds and compositions of the present invention will bedecided by the attending physician within the scope of sound medicaljudgment. The specific therapeutically effective dose level for anyparticular patient will depend upon a variety of factors including thedisorder being treated and the severity of the brain injury or ischemia;activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coinciding with the specificcompound employed; and like factors well known in the medical arts.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell assays or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell based assays. A dose may be formulatedin animal models to achieve a circulating plasma concentration rangethat includes the IC50 (i.e., the concentration of the test compoundwhich achieves a half-maximal inhibition of symptoms) as determined incell culture. Such information can be used to more accurately determineuseful doses in humans. Levels in plasma may be measured, for example,by high performance liquid chromatography.

The total daily dose of the active compounds of the present inventionadministered to a subject in single or in divided doses can be inamounts, for example, from 0.01 to 25 mg/kg body weight or more usuallyfrom 0.1 to 15 mg/kg body weight. Single dose compositions may containsuch amounts or submultiples thereof to make up the daily dose. Ingeneral, treatment regimens according to the present invention compriseadministration to a human or other mammal in need of such treatment fromabout 1 mg to about 1000 mg of the active substance(s) of this inventionper day in multiple doses or in a single dose of from 1 mg, 5 mg, 10 mg,100 mg, 500 mg or 1000 mg.

In certain situations, it may be important to maintain a fairly highdose of the active agent in the blood stream of the patient,particularly early in the treatment. Such a fairly high dose may includea dose that is several times greater than its use in other indications.For example, the typical anti-diabetic dose of oral or IV glibenclamideis about 2.5 mg/kg to about 15 mg/kg per day; the typical anti-diabeticdose of oral or IV tolbutamide is about to 0.5 gm/kg to about 2.0 gm/kgper day; the typical anti-diabetic dose for oral gliclazide is about 30mg/kg to about 120 mg/kg per day; however, much larger doses may berequired to block neural cell swelling and brain swelling.

For example, in one embodiment of the present invention directed to amethod of preventing neuronal cell swelling in the brain of a subject byadministering to the subject a formulation containing an effectiveamount of a compound that blocks the NC_(Ca-ATP) channel and apharmaceutically acceptable carrier; such formulations may contain fromabout 0.1 to about 100 grams of tolbutamide or from about 0.5 to about150 milligrams of glibenclamide. In another embodiment of the presentinvention directed to a method of alleviating the negative effects oftraumatic brain injury or cerebral ischemia stemming from neural cellswelling in a subject by administering to the subject a formulationcontaining an effective amount of a compound that blocks the NC_(Ca-ATP)channel and a pharmaceutically acceptable carrier.

In situations of traumatic brain injury or cerebral ischemia (such asstroke), or cerebral hypoxia, it may be important to maintain a fairlyhigh dose of the active agent to ensure delivery to the brain of thepatient, particularly early in the treatment. Hence, at least initially,it may be important to keep the dose relatively high and/or at asubstantially constant level for a given period of time, preferably, atleast about six or more hours, more preferably, at least about twelve ormore hours and, most preferably, at least about twenty-four or morehours. In situations of traumatic brain injury or cerebral ischemia(such as stroke), it may be important to maintain a fairly high dose ofthe active agent to ensure delivery to the brain of the patient,particularly early in the treatment.

When the method of the present invention is employed to treat conditionsinvolving bleeding in the brain, such as traumatic brain injury orcerebral ischemia (such as stroke), delivery via the vascular system isavailable and the compound is not necessarily required to readily crossthe blood-brain barrier.

C. Formulations and Administration

The compounds of the present invention may be administered alone or incombination or in concurrent therapy with other agents which affect thecentral or peripheral nervous system, particularly selected areas of thebrain.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs containing inert diluents commonly used in the art, such aswater, isotonic solutions, or saline. Such compositions may alsocomprise adjuvants, such as wetting agents; emulsifying and suspendingagents; sweetening, flavoring and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulation can be sterilized, for example, by filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions, which can be dissolvedor dispersed in sterile water or other sterile injectable medium justprior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of a drug from subcutaneous or intramuscular injection.The most common way to accomplish this is to inject a suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug becomes dependent on the rate of dissolutionof the drug, which is, in turn, dependent on the physical state of thedrug, for example, the crystal size and the crystalline form. Anotherapproach to delaying absorption of a drug is to administer the drug as asolution or suspension in oil. Injectable depot forms can also be madeby forming microcapsule matrices of drugs and biodegradable polymers,such as polylactide-polyglycoside. Depending on the ratio of drug topolymer and the composition of the polymer, the rate of drug release canbe controlled. Examples of other biodegradable polymers includepolyorthoesters and polyanhydrides. The depot injectables can also bemade by entrapping the drug in liposomes or microemulsions, which arecompatible with body tissues.

Suppositories for rectal administration of the drug can be prepared bymixing the drug with a suitable non-irritating excipient, such as cocoabutter and polyethylene glycol which are solid at ordinary temperaturebut liquid at the rectal temperature and will, therefore, melt in therectum and release the drug.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, gelcaps and granules. In such solid dosageforms the active compound may be admixed with at least one inert diluentsuch as sucrose, lactose or starch. Such dosage forms may also comprise,as is normal practice, additional substances other than inert diluents,e.g., tableting lubricants and other tableting aids such as magnesiumstearate and microcrystalline cellulose. In the case of capsules,tablets and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings andother release-controlling coatings.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,capsules, pills, and granules can be prepared with coatings and shellssuch as enteric coatings and other coatings well known in thepharmaceutical formulating art. They may optionally contain opacifyingagents and can also be of a composition that they release the activeingredient(s) only, or preferably, in a certain part of the intestinaltract, optionally in a delayed manner Examples of embedding compositionswhich can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention further include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. Transdermal patcheshave the added advantage of providing controlled delivery of activecompound to the body. Such dosage forms can be made by dissolving ordispersing the compound in the proper medium. Absorption enhancers canalso be used to increase the flux of the compound across the skin. Therate can be controlled by either providing a rate controlling membraneor by dispersing the compound in a polymer matrix or gel. The ointments,pastes, creams and gels may contain, in addition to an active compoundof this invention, excipients such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

The method of the present invention employs the compounds identifiedherein for both in vitro and in vivo applications. For in vivoapplications, the invention compounds can be incorporated into apharmaceutically acceptable formulation for administration. Those ofskill in the art can readily determine suitable dosage levels when theinvention compounds are so used.

As employed herein, the phrase “suitable dosage levels” refers to levelsof compound sufficient to provide circulating concentrations high enoughto effectively block the NC_(Ca-ATP) channel and prevent or reduceneural cell swelling in vivo.

In accordance with a particular embodiment of the present invention,compositions comprising at least one SUR1 antagonist compound (asdescribed above), and a pharmaceutically acceptable carrier arecontemplated.

Exemplary pharmaceutically acceptable carriers include carriers suitablefor oral, intravenous, subcutaneous, intramuscular, intracutaneous, andthe like administration. Administration in the form of creams, lotions,tablets, dispersible powders, granules, syrups, elixirs, sterile aqueousor non-aqueous solutions, suspensions or emulsions, and the like, iscontemplated.

For the preparation of oral liquids, suitable carriers includeemulsions, solutions, suspensions, syrups, and the like, optionallycontaining additives such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring and perfuming agents, and the like.

For the preparation of fluids for parenteral administration, suitablecarriers include sterile aqueous or non-aqueous solutions, suspensions,or emulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They may be sterilized, for example,by filtration through a bacteria-retaining filter, by incorporatingsterilizing agents into the compositions, by irradiating thecompositions, or by heating the compositions. They can also bemanufactured in the form of sterile water, or some other sterileinjectable medium immediately before use. The active compound is admixedunder sterile conditions with a pharmaceutically acceptable carrier andany needed preservatives or buffers as may be required.

The treatments may include various “unit doses.” Unit dose is defined ascontaining a predetermined quantity of the therapeutic composition (anantagonist of the NC_(Ca-ATP) channel or its related-compounds thereof)calculated to produce the desired responses in association with itsadministration, e.g., the appropriate route and treatment regimen. Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. Also of import isthe subject to be treated, in particular, the state of the subject andthe protection desired. A unit dose need not be administered as a singleinjection but may comprise continuous infusion over a set period oftime.

X. COMBINATION TREATMENTS

In the context of the present invention, it is contemplated that anantagonist of the NC_(Ca-ATP) channel or related-compounds thereof maybe used in combination with an additional therapeutic agent to moreeffectively treat a cerebral ischemic event, and/or decreaseintracranial pressure. In some embodiments, it is contemplated that aconventional therapy or agent, including but not limited to, apharmacological therapeutic agent may be combined with the antagonist orrelated-compound of the present invention.

Pharmacological therapeutic agents and methods of administration,dosages, etc. are well known to those of skill in the art (see forexample, the “Physicians Desk Reference”, Goodman & Gilman's “ThePharmacological Basis of Therapeutics”, “Remington's PharmaceuticalSciences”, and “The Merck Index, Eleventh Edition”, incorporated hereinby reference in relevant parts), and may be combined with the inventionin light of the disclosures herein. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject, and suchindividual determinations are within the skill of those of ordinaryskill in the art.

Non-limiting examples of a pharmacological therapeutic agent that may beused in the present invention include an antihyperlipoproteinemic agent,an antiarteriosclerotic agent, an anticholesterol agent, anantiinflammatory agent, an antithrombotic/fibrinolytic agent,anticoagulant, antiplatelet, vasodilator, and/or diuretics.Thromoblytics that are used can include, but are not limited toprourokinase, streptokinase, and tissue plasminogen activator (tPA)Anticholesterol agents include but are not limited to HMG-CoA Reductaseinhibitors, cholesterol absorption inhibitors, bile acid sequestrants,nicotinic acid and derivatives thereof, fibric acid and derivativesthereof. HMG-CoA Reductase inhibitors include statins, for example, butnot limited to atorvastatin calcium (Lipitor®), cerivastatin sodium(Baycol®), fluvastatin sodium (Lescol®), lovastatin (Advicor®),pravastatin sodium (Pravachol®), and simvastatin (Zocor®). Agents knownto reduce the absorption of ingested cholesterol include, for example,Zetia®. Bile acid sequestrants include, but are not limited tocholestryramine, cholestipol and colesevalam. Other anticholesterolagents include fibric acids and derivatives thereof (e.g., gemfibrozil,fenofibrate and clofibrate); nicotinic acids and derivatives thereof(e.g., nician, lovastatin) and agents that extend the release ofnicotinic acid, for example niaspan. Antiinflammatory agents include,but are not limited to non-sterodial anti-inflammatory agents (e.g.,naproxen, ibuprofen, celeoxib) and sterodial anti-inflammatory agents(e.g., glucocorticoids). Anticoagulants include, but are not limited toheparin, warfarin, and coumadin. Antiplatelets include, but are notlimited to aspirin, and aspirin related-compounds, for exampleacetaminophen. Diuretics include, but are not limited to such asfurosemide (Lasix®), bumetanide (Bumex®), torsemide (Demadex®), thiazide& thiazide-like diuretics (e.g., chlorothiazide (Diuril®) andhydrochlorothiazide (Esidrix®), benzthiazide, cyclothiazide, indapamide,chlorthalidone, bendroflumethizide, metolazone), amiloride, triamterene,and spironolacton. Vasodilators include, but are not limited tonitroglycerin.

Thus, in certain embodiments, the present invention comprisesco-administration of an antagonist of the NC_(Ca-ATP) channel with athrombolytic agent. Co-administration of these two compounds willincrease the therapeutic window of the thrombolytic agent. Examples ofsuitable thrombolytic agents that can be employed in the methods andpharmaceutical compositions of this invention are prourokinase,streptokinase, and tissue plasminogen activator (tPA).

In certain embodiments, the present invention comprisesco-administration of an antagonist of the NC_(Ca-ATP) channel withglucose or related carbohydrate to maintain appropriate levels of serumglucose. Appropriate levels of blood glucose are within the range ofabout 60 mmol/l to about 150 mmol/liter. Thus, glucose or a relatedcarbohydrate is administered in combination to maintain the serumglucose within this range.

When an additional therapeutic agent, as long as the dose of theadditional therapeutic agent does not exceed previously quoted toxicitylevels, the effective amounts of the additional therapeutic agent maysimply be defined as that amount effective to reduce cerebral edema whenadministered to an animal in combination with an agonist of NC_(Ca-ATP)channel or related-compounds thereof. This may be easily determined bymonitoring the animal or patient and measuring those physical andbiochemical parameters of health and disease that are indicative of thesuccess of a given treatment. Such methods are routine in animal testingand clinical practice.

To inhibit hemorrhagic conversion, reduce cell swelling, etc., using themethods and compositions of the present invention, one would generallycontact a cell with antagonist of NC_(Ca-ATP) channel orrelated-compounds thereof in combination with an additional therapeuticagent, such as tPA, aspirin, statins, diuretics, warfarin, coumadin,mannitol, etc. These compositions would be provided in a combined amounteffective to inhibit hemorrhagic conversion, cell swelling and edema.This process may involve contacting the cells with agonist ofNC_(Ca-ATP) channel or related-compounds thereof in combination with anadditional therapeutic agent or factor(s) at the same time. This may beachieved by contacting the cell with a single composition orpharmacological formulation that includes both agents, or by contactingthe cell with two distinct compositions or formulations, at the sametime, wherein one composition includes an antagonist of the NC_(Ca-ATP)channel or derivatives thereof and the other includes the additionalagent.

Alternatively, treatment with an antagonist of NC_(Ca-ATP) channel orrelated-compounds thereof may precede or follow the additional agenttreatment by intervals ranging from minutes to hours to weeks to months.In embodiments where the additional agent is applied separately to thecell, one would generally ensure that a significant period of time didnot expire between the time of each delivery, such that the agent wouldstill be able to exert an advantageously combined effect on the cell. Insuch instances, it is contemplated that one would contact the cell withboth modalities within about 1-24 hr of each other and, more preferably,within about 6-12 hr of each other.

Typically, for maximum benefit of the thrombolytic agent, or therapymust be started within three hours of the onset of stroke symptoms,making rapid diagnosis and differentiation of stroke and stroke typecritical. However, in the present invention, administration of theNC_(Ca-ATP) channel with a thrombolytic agent increases this therapeuticwindow. The therapeutic window for thrombolytic agents may be increasedby several (4-8) hours by co-administering antagonist of the NC_(Ca-ATP)channel.

Further embodiments include treatment with SUR1 antagonist, thrombolyticagent, and glucose. Glucose administration may be at the time oftreatment with SUR1 antagonist, or may follow treatment with SUR1antagonist (e.g., at 15 minutes after treatment with SUR1 antagonist, orat one half hour after treatment with SUR1 antagonist, or at one hourafter treatment with SUR1 antagonist, or at two hours after treatmentwith SUR1 antagonist, or at three hours after treatment with SUR1antagonist). Glucose administration may be by intravenous, orintraperitoneal, or other suitable route and means of delivery.Additional glucose allows administration of higher doses of SUR1antagonist than might otherwise be possible. Treatment with glucose inconjunction with treatment with SUR1 antagonist (at the same time astreatment with SUR1 antagonist, or at a later time after treatment withSUR1 antagonist) may further enlarge the time window after stroke,trauma, or other brain injury when thrombolytic treatment may beinitiated.

Yet further, the combination of the antagonist and tPA results in adecrease or prevention of hemorrhagic conversion following reperfusion.Hemorrhagic conversion is the transformation of a bland infarct into ahemorrhagic infarct after restoration of circulation. It is generallyaccepted that these complications of stroke and of reperfusion areattributable to capillary endothelial cell dysfunction that worsens asischemia progresses. Thus, the present invention is protective of theendothelial cell dysfunction that occurs as a result of an ischemicevent.

Endothelial cell dysfunction comprises three phases. Phase one ischaracterized by formation of ionic edema with the blood brain barrierstill intact. The second phase is characterized by formation ofvasogenic edema in which the blood brain barrier is no longer intact.Phase three is characterized by hemorrhagic conversion due to failure ofcapillary integrity during which all constituents of blood, includingerythrocytes, extravasate into brain parenchyma. Disruption of BBBinvolves ischemia-induced activation of endothelial cells that resultsin expression and release of MMPs, specifically, MMP-2 (gelatinase A)and MMP-9 (gelatinase B).

Since hemorrhagic conversion increases mortality of the patient, it isessential that these patients receive treatment in an urgent manner. Forexample, it is known that hemorrhagic conversion typically results inpatients if reperfusion and tPA treatment is delayed beyond 3 hr or moreafter thrombotic stroke. Thus, the administration of the antagonist ofthe present invention will reduce necrotic death of ischemic endothelialcells, and will thereby prolong the therapeutic window for tPA, therebydecreasing mortality of the patient.

XI. DIAGNOSTICS

The antagonist or related-compound can be used for diagnosing,monitoring, or prognosis of ischemia or damage to neurons, glial cellsor in monitoring neuronal cells in zones of cerebral edema, metastatictumors, etc.

A. Genetic Diagnosis

One embodiment of the instant invention comprises a method for detectingexpression of any portion of a Na_(Ca-ATP) channel, for example,expression of the regulatory unit, SUR1, and/or expression of thepore-forming subunit. This may comprise determining the level of SUR1expressed and/or the level of the pore-forming subunit expressed. It isunderstood by the present invention that the up-regulation or increasedexpression of the Na_(Ca-ATP) channel relates to increased levels ofSUR1, which correlates to increased neuronal damage, such as cerebraledema.

Firstly, a biological sample is obtained from a subject. The biologicalsample may be tissue or fluid. In certain embodiments, the biologicalsample includes cells from the brain and/or cerebral endothelial cellsor microvessels and/or gliotic capsule. For example, in metastatictumors, glial cells are activated and form a capsule around the tumor.

Nucleic acids used are isolated from cells contained in the biologicalsample, according to standard methodologies (Sambrook et al., 1989). Thenucleic acid may be genomic DNA or fractionated or whole cell RNA. WhereRNA is used, it may be desired to convert the RNA to a complementary DNA(cDNA). In one embodiment, the RNA is whole cell RNA; in another, it ispoly-A RNA. Normally, the nucleic acid is amplified.

Depending on the format, the specific nucleic acid of interest isidentified in the sample directly using amplification or with a second,known nucleic acid following amplification. Next, the identified productis detected. In certain applications, the detection may be performed byvisual means (e.g., ethidium bromide staining of a gel). Alternatively,the detection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of radiolabel or fluorescentlabel or even via a system using electrical or thermal impulse signals(Affymax Technology; Bellus, 1994).

Following detection, one may compare the results seen in a given subjectwith a statistically significant reference group of normal subjects andsubjects that have been diagnosed with a stroke, cancer, cerebral edema,etc.

Yet further, it is contemplated that chip-based DNA technologies such asthose described by Hacia et al., (1996) and Shoemaker et al., (1996) canbe used for diagnosis. Briefly, these techniques involve quantitativemethods for analyzing large numbers of genes rapidly and accurately. Bytagging genes with oligonucleotides or using fixed probe arrays, one canemploy chip technology to segregate target molecules as high densityarrays and screen these molecules on the basis of hybridization. Seealso Pease et al., (1994); Fodor et al., (1991).

B. Other Types of Diagnosis

In order to increase the efficacy of molecules, for example, compoundsand/or proteins and/or antibodies, as diagnostic agents, it isconventional to link or covalently bind or complex at least one desiredmolecule or moiety.

Certain examples of conjugates are those conjugates in which themolecule (for example, protein, antibody, and/or compound) is linked toa detectable label. “Detectable labels” are compounds and/or elementsthat can be detected due to their specific functional properties, and/orchemical characteristics, the use of which allows the antibody to whichthey are attached to be detected, and/or further quantified if desired.

Conjugates are generally preferred for use as diagnostic agents.Diagnostics generally fall within two classes, those for use in in vitrodiagnostics, such as in a variety of immunoassays, and/or those for usein vivo diagnostic protocols, generally known as “molecule-directedimaging”.

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to molecules, for example, antibodies (see, for e.g.,U.S. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporatedherein by reference). The imaging moieties used can be paramagneticions; radioactive isotopes; fluorochromes; NMR-detectable substances;X-ray imaging.

In the case of paramagnetic ions, one might mention by way of exampleions such as chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might mention ²¹¹astatine, ¹¹carbon, ¹⁴carbon,⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, ⁶⁷copper, ¹⁵²Eu, ⁶⁷gallium,³hydrogen ¹²³iodine, ¹²⁵iodine, ¹³¹iodine, ¹¹¹indium, ⁵²iron,³²phosphorus, ¹⁸⁶rhenium, ¹⁸⁸rhenium, ⁷⁵selenium, ³⁵sulphur,^(99m)technicium and/or ⁹⁰yttrium. ¹²⁵I is often being preferred for usein certain embodiments, and ^(99m) technicium and/or ¹¹¹indium are alsooften preferred due to their low energy and suitability for long rangedetection.

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed.

Another type of conjugates contemplated in the present invention arethose intended primarily for use in vitro, where the molecule is linkedto a secondary binding ligand and/or to an enzyme (an enzyme tag) thatwill generate a colored product upon contact with a chromogenicsubstrate. Examples of suitable enzymes include urease, alkalinephosphatase, (horseradish) hydrogen peroxidase or glucose oxidase.Preferred secondary binding ligands are biotin and/or avidin andstreptavidin compounds. The use of such labels is well known to those ofskill in the art and are described, for example, in U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and4,366,241; each incorporated herein by reference.

The steps of various other useful immunodetection methods have beendescribed in the scientific literature, such as, e.g., Nakamura et al.,(1987) Immunoassays, in their most simple and direct sense, are bindingassays. Certain preferred immunoassays are the various types ofradioimmunoassays (RIA) and immunobead capture assay Immunohistochemicaldetection using tissue sections also is particularly useful. However, itwill be readily appreciated that detection is not limited to suchtechniques, and Western blotting, dot blotting, FACS analyses, and thelike also may be used in connection with the present invention.

Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary molecules/antibodies against the SUR1 orregulatory subunit of the NC_(Ca-ATP) channel are considered to be ofparticular use in this regard. U.S. patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody or abiotin/avidin ligand binding arrangement, as is known in the art.

In addition to the above imaging techniques, one of skill in the art isalso aware that positron emission tomography, PET imaging or a PET scan,can also be used as a diagnostic examination. PET scans involve theacquisition of physiologic images based on the detection of radiationfrom the emission of positrons. Positrons are tiny particles emittedfrom a radioactive substance administered to the subject.

Thus, in certain embodiments of the present invention, the antagonist orrelated-compound thereof is enzymatically-, radiolabel-, orfluorescently-tagged, as described above and used to diagnosis, monitor,and/or stage neuronal damage by cerebral edema. For example, theenzymatically-, radiolabel-, or fluorescently-tagged antagonist orrelated-compound thereof can be used to determine the size, limitsand/or boundaries of tumors. It is difficult to determine the boundariesof certain tumors, for example, metastatic tumors. In metastatic tumors,glial cells are activated and form a capsule or gliotic capsule aroundthe tumor. Thus, the labeled antagonist or related-compound thereof canbe used to determine the border of tumor, which can enhance theefficiency of its removal by the surgeon. Still further, the labeledantagonist or related-compound thereof may be used to determine ordefine the penumbra or the areas at risk for later infarction or damageafter a stroke.

C. Formulations and Routes for Administration of Compounds

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more modulators of NC_(Ca-ATP) channel(antagonist and/or agonist) or related-compounds or additional agentdissolved or dispersed in a pharmaceutically acceptable carrier. Thephrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, suchas, for example, a human, as appropriate. The preparation of apharmaceutical composition that contains at least one modulators ofNC_(Ca-ATP) channel (antagonist and/or agonist) or related-compounds oradditional active ingredient will be known to those of skill in the artin light of the present disclosure, as exemplified by Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,incorporated herein by reference. Moreover, for animal (e.g., human)administration, it will be understood that preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

The modulators of NC_(Ca-ATP) channel (antagonist and/or agonist) orrelated-compounds may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. The present invention can be administered intravenously,intradermally, transdermally, intrathecally, intraventricularly,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, topically, intramuscularly, subcutaneously, mucosally,orally, topically, locally, inhalation (e.g., aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in cremes, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art (see,for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack PrintingCompany, 1990, incorporated herein by reference).

The modulators of NC_(Ca-ATP) channel (antagonist and/or agonist) orrelated-compounds may be formulated into a composition in a free base,neutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as formulated for parenteraladministrations such as injectable solutions, or aerosols for deliveryto the lungs, or formulated for alimentary administrations such as drugrelease capsules and the like.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of the composition contained therein, its usein administrable composition for use in practicing the methods of thepresent invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include modulators ofNC_(Ca-ATP) channel (antagonist and/or agonist) or related-compounds,one or more lipids, and an aqueous solvent. As used herein, the term“lipid” will be defined to include any of a broad range of substancesthat is characteristically insoluble in water and extractable with anorganic solvent. This broad class of compounds is well known to those ofskill in the art, and as the term “lipid” is used herein, it is notlimited to any particular structure. Examples include compounds whichcontain long-chain aliphatic hydrocarbons and their derivatives. A lipidmay be naturally occurring or synthetic (i.e., designed or produced byman). However, a lipid is usually a biological substance. Biologicallipids are well known in the art, and include for example, neutral fats,phospholipids, phosphoglycerides, steroids, terpenes, lysolipids,glycosphingolipids, glycolipids, sulphatides, lipids with ether andester-linked fatty acids and polymerizable lipids, and combinationsthereof. Of course, compounds other than those specifically describedherein that are understood by one of skill in the art as lipids are alsoencompassed by the compositions and methods of the present invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the modulators of NC_(Ca-ATP) channel (antagonistand/or agonist) or related-compounds may be dispersed in a solutioncontaining a lipid, dissolved with a lipid, emulsified with a lipid,mixed with a lipid, combined with a lipid, covalently bonded to a lipid,contained as a suspension in a lipid, contained or complexed with amicelle or liposome, or otherwise associated with a lipid or lipidstructure by any means known to those of ordinary skill in the art. Thedispersion may or may not result in the formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeutic and/orprophylatic interventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

Pharmaceutical formulations may be administered by any suitable route ormeans, including alimentary, parenteral, topical, mucosal or other routeor means of administration. Alimentary routes of administration includeadministration oral, buccal, rectal and sublingual routes. Parenteralroutes of administration include administration include injection intothe brain parenchyma, and intravenous, intradermal, intramuscular,intraarterial, intrathecal, subcutaneous, intraperitoneal, andintraventricular routes of administration. Topical routes ofadministration include transdermal administration.

D. Alimentary Compositions and Formulations

In preferred embodiments of the present invention, the modulators ofNC_(Ca-ATP) channel (antagonist and/or agonist) or related-compounds areformulated to be administered via an alimentary route. Alimentary routesinclude all possible routes of administration in which the compositionis in direct contact with the alimentary tract. Specifically, thepharmaceutical compositions disclosed herein may be administered orally,buccally, rectally, or sublingually. As such, these compositions may beformulated with an inert diluent or with an assimilable edible carrier,or they may be enclosed in hard- or soft-shell gelatin capsule, or theymay be compressed into tablets, or they may be incorporated directlywith the food of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515;5,580,579 and 5,792, 451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, such as, for example,gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof; a disintegrating agent, such as, forexample, corn starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier. Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup of elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

Additional formulations which are suitable for other modes of alimentaryadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum. After insertion, suppositories soften, melt or dissolvein the cavity fluids. In general, for suppositories, traditionalcarriers may include, for example, polyalkylene glycols, triglyceridesor combinations thereof. In certain embodiments, suppositories may beformed from mixtures containing, for example, the active ingredient inthe range of about 0.5% to about 10%, and preferably about 1% to about2%.

E. Parenteral Compositions and Formulations

In further embodiments, modulators of NC_(Ca-ATP) channel (antagonistand/or agonist) or related-compounds may be administered via aparenteral route. As used herein, the term “parenteral” includes routesthat bypass the alimentary tract. Specifically, the pharmaceuticalcompositions disclosed herein may be administered for example, but notlimited to intravenously, intradermally, intramuscularly,intraarterially, intraventricularly, intrathecally, subcutaneous, orintraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308, 5,466,468,5,543,158; 5,641,515; and 5,399,363 (each specifically incorporatedherein by reference in its entirety).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, DMSO, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

F. Miscellaneous Pharmaceutical Compositions and Formulations

In other preferred embodiments of the invention, the active compoundmodulators of NC_(CaATP) channel (antagonist and/or agonist) orrelated-compounds may be formulated for administration via variousmiscellaneous routes, for example, topical (i.e., transdermal)administration, mucosal administration (intranasal, vaginal, etc.)and/or inhalation.

Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-solubly based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andluarocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch”. For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays has been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

XII. DIAGNOSTIC OR THERAPEUTIC KITS

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, it is envisioned that a compound that selectivelybinds to or identifies SUR1 may be comprised in a diagnostic kit. Suchcompounds can be referred to as an “SUR1 marker”, which may include, butare not limited to antibodies (monoclonal or polyclonal), SUR1oligonucleotides, SUR1 polypeptides, small molecule or combinationsthereof, antagonist, agonist, etc. It is envisioned that any of theseSUR1 markers may be linked to a radioactive substance and/or afluorescent marker and/or a enzymatic tag for quick determination. Thekits may also comprise, in suitable container means a lipid, and/or anadditional agent, for example a radioactive or enzymatic or florescentmarker.

The kits may comprise a suitably aliquoted SUR1 marker, lipid and/oradditional agent compositions of the present invention, whether labeledor unlabeled, as may be used to prepare a standard curve for a detectionassay. The components of the kits may be packaged either in aqueousmedia or in lyophilized form. The container means of the kits willgenerally include at least one vial, test tube, flask, bottle, syringeor other container means, into which a component may be placed, andpreferably, suitably aliquoted. Where there are more than one componentin the kit, the kit also will generally contain a second, third or otheradditional container into which the additional components may beseparately placed. However, various combinations of components may becomprised in a vial. The kits of the present invention also willtypically include a means for containing the SUR1 marker, lipid,additional agent, and any other reagent containers in close confinementfor commercial sale. Such containers may include injection or blowmolded plastic containers into which the desired vials are retained.

Therapeutic kits of the present invention are kits comprising anantagonist, agonist or an related-compound thereof. Depending upon thecondition and/or disease that is being treated, the kit may comprise anSUR1 antagonist or related-compound thereof to block and/or inhibit theNC_(Ca-ATP) channel or the kit may comprise an SUR1 agonist orrelated-compound thereof to open the NC_(Ca-ATP) channel. Such kits willgenerally contain, in suitable container means, a pharmaceuticallyacceptable formulation of SUR1 antagonist, agonist or related-compoundthereof. The kit may have a single container means, and/or it may havedistinct container means for each compound. For example, the therapeuticcompound and solution may be contained within the same container;alternatively, the therapeutic compound and solution may each becontained within different containers. A kit may include a containerwith the therapeutic compound that is contained within a container ofsolution.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The SUR1 antagonist,agonist or related-compounds thereof may also be formulated into asyringeable composition. In which case, the container means may itselfbe a syringe, pipette, and/or other such like apparatus, from which theformulation may be applied to an infected area of the body, injectedinto an animal, and/or even applied to and/or mixed with the othercomponents of the kit.

Examples of aqueous solutions include, but are not limited to ethanol,DMSO and/or Ringer's solution. In certain embodiments, the concentrationof DMSO or ethanol that is used is no greater than 0.1% or (1 ml/1000L).

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

The container means will generally include at least one vial, test tube,flask, bottle, syringe and/or other container means, into which the SUR1antagonist, agonist or related-compounds thereof is suitably allocated.The kits may also comprise a second container means for containing asterile, pharmaceutically acceptable buffer and/or other diluent.

The kits of the present invention will also typically include a meansfor containing the vials in close confinement for commercial sale, suchas, e.g., injection and/or blow-molded plastic containers into which thedesired vials are retained.

Irrespective of the number and/or type of containers, the kits of theinvention may also comprise, and/or be packaged with, an instrument forassisting with the injection/administration and/or placement of the SUR1antagonist, agonist or related-compounds thereof within the body of ananimal. Such an instrument may be a syringe, pipette, forceps, and/orany such medically approved delivery vehicle.

In addition to the SUR1 antagonist, agonist or related-compoundsthereof, the kits may also include a second active ingredient. Examplesof the second active ingredient include substances to preventhypoglycemia (e.g., glucose, D5W, glucagon, etc.), thrombolytic agents,anticoagulants, antiplatelets, statins, diuretics, vasodilators, etc.These second active ingredients may be combined in the same vial as theSUR1 antagonist, agonist or related-compounds thereof or they may becontained in a separate vial.

Still further, the kits of the present invention can also includeglucose testing kits. Thus, the blood glucose of the patient is measuredusing the glucose testing kit, then the SUR1 antagonist, agonist orrelated-compounds thereof can be administered to the subject followed bymeasuring the blood glucose of the patient.

In addition to the above kits, the therapeutic kits of the presentinvention can be assembled such that an IV bag comprises a septum orchamber which can be opened or broken to release the compound into theIV bag. Another type of kit may include a bolus kit in which the boluskit comprises a pre-loaded syringe or similar easy to use, rapidlyadministrable device. An infusion kit may comprise the vials or ampoulesand an IV solution (e.g., Ringer's solution) for the vials or ampoulesto be added prior to infusion. The infusion kit may also comprise abolus kit for a bolus/loading dose to be administered to the subjectprior, during or after the infusion.

EXAMPLES

The following examples are provided for further illustration of thepresent invention, and do not limit the invention. The examples providedherein are for illustrative purposes only, and are in no way intended tolimit the scope of the present invention. While the invention has beendescribed in detail, and with reference to specific embodiments thereof,it will be apparent to one with ordinary skill in the art that variouschanges and modifications can be made in the specific embodiments whichare disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention. Experiments andexemplary procedures are described below which provide additionalenabling support for the present invention. In particular, in vitrostudies using freshly isolated reactive astrocytes and in vivo studiesusing appropriate animal models are disclosed.

Cell Preparation

Reactive astrocytes are produced in vivo and harvested from adult brainin the following manner gelatin sponges (Gelfoam®, Upjohn Co., KalamazooMich.) are implanted into a stab wound in the parietal lobe of 8 weekold Wistar rats as described herein. Sponge pieces are harvested at 8days and washed three times in phosphate-buffered saline (PBS, pH 7.4)to remove adherent tissue. Depending on the number of NRAs required fora particular study, the sponge pieces may be harvested earlier or laterafter implantation into a stab wound, with the preferred harvest beingconducted from about 2 days to about 30 days after implantation, and themost preferred range being conducted from about 2 days to about 3 daysafter implantation.

NRAs are freshly isolated from the sponge pieces in the followingmanner: washed pieces are placed in an Eppendorf tube containingartificial cerebrospinal fluid (aCSF) composed of (mM): 124 mM NaCl, 5.0mM, 1.3 mM MgCl₂, 2.0 mM CaCl₂, 26 mM NaHCO₃, and 10 mM D-glucose; at pH7.4, ≈290 mOsm, wherein the aCSF contains papain 20 U/ml, trypsininhibitor 10 mg/ml and DNase 0.01% (Worthington, Lakewood, N.J.), theentirety of which is referred to as a “digestion system.”

This digestion system is transferred to an incubator (humidified 90%/10%air/CO2, 37° C.) for 20 minutes, and is gently triturated every 5minutes. The cell suspension is centrifuged at 3,000 rpm for 1 minute.The pelleted cells are resuspended in aCSF and stored at 4° C. untilstudied.

For some studies, prior to resuspension in aCSF, the pelleted cells canbe further purified by removing red blood cells (RBCs) using densitygradient centrifugation in Histopaque-1 077 (Sigma Diagnostics, St.Louis, Mo.). This further purification process can produce a populationof cells in which <<1% are RBCs, as determined by phase contrastmicroscopy.

Scanning Electron Microscopy (SEM)

To study cell blebbing and swelling, freshly isolated cells are exposedat room temperature to NaN₃ then, after various time intervals, cellsare fixed using iced 4% formaldehyde+1% glutaraldehyde for 24 hours thendehydrated using serial concentrations (35, 50, 75, 95, 100%) ofethanol. Specimens are critical point dried (Tousimis), gold coated(Technics), and viewed using an AMR 1000 scanning electron microscope.

Electrophysiology

Experiments are carried out at room temperature, 22-25° C., using NRAswithin 24 hour of cell isolation. An aliquot of these freshly isolatedNRAs is placed in the recording chamber filled with extracellular bathsolution containing (a): NaCl 130, KCl 10, CaCl₂ 1, MgCl₂ 1, HEPES 32.5,glucose 12.5, pH 7.4. After viable cells adhere to the surface, flushingwith excess solution washes away residual debris not previously removedby centrifugation. Membrane currents are amplified (Axopatch 200A, AxonInstruments, Foster City, Calif.) and sampled on-line at 5 kHz using amicrocomputer equipped with a digitizing board (Digidata 1200A, AxonInstruments) and running Clampex software (version 8.0, AxonInstruments). Membrane currents are recorded in intact cells using boththe cell-attached and the nystatin-perforated whole-cell configurations,according to methods described in Horn and Marty, 1988. Membranecurrents are recorded in cell-free isolated membrane patches, using boththe inside-out and outside-out configurations, such as those describedin Hamill et al., 1981. Patch clamp pipettes, pulled from borosilicateglass (Kimax, Fisher Scientific, Pittsburgh, Pa.), have resistances of6-8 MΩ a for single channel recordings and 2-4 MΩ a for experimentsusing the nystatin-perforated whole-cell technique. The bath electrodeis a Ag/AgCl pellet (Clark Electromedical, Reading, England) that isplaced directly in the bath except when the bath [Cl−] is altered, inwhich case an agar bridge made with 3 M KCl is used to connect to thebath.

The terms “intracellular” and “cytoplasmic” are interchangeable, as arethe terms “extracellular” and “external”. The terms “voltage” and“potential” are interchangeable when referring to membrane voltage ormembrane potential. “Clamping” a cell membrane refers to holding thevoltage across the cell membrane constant and measuring changes inmembrane current as membrane resistance changes due to ion channelopening and closing (“voltage clamp”) or holding the current across thecell membrane constant and measuring changes in membrane voltage asmembrane resistance changes due to ion channel opening and closing(“current clamp”). When a membrane voltage is imposed on the cell, forexample with a “ramp” or “pulse”, it is understood that the cellmembrane has been voltage-clamped and membrane current is beingmeasured. When membrane “resting potential” is measured, it isunderstood that the cell membrane has been current-clamped and membranevoltage is being measured.

The “whole-cell” experimental configuration refers to a situation inwhich a recording pipette penetrates the cell membrane so that thepipette solution is continuous with the cytoplasm or the membrane underthe pipette is perforated using nystatin, the external solution is incontact with the extracellular membrane, and current or voltagerecordings represent measurements from the entire cell membrane. The“cell-attached patch” experimental configuration refers to a situationin which the pipette contacts the cell so that the patch is stillforming part of the intact cell membrane and channels in the patch arerecorded. The “outside-out patch” experimental configuration refers to asituation in which an excised patch of cell membrane is sealed to thetip of a recording pipette so that the pipette solution is in contactwith the extracellular side of the membrane, the external solution is incontact with the cytoplasmic side of the membrane, and current orvoltage recordings represent measurements from the excised patch ofmembrane. The “inside-out patch” experimental configuration refers to asituation in which an excised patch of cell membrane is sealed to thetip of a recording pipette so that the pipette solution is in contactwith the cytoplasmic side of the membrane, the external solution is incontact with the extracellular side of the membrane, and current orvoltage recordings represent measurements from the excised patch ofmembrane.

The term “patches” includes, but is not limited to: inside-out patches,outside-out patches, an excised patch of a cell membrane, or acell-attached patch. The term “membrane preparation” includes patches aswell as cell membranes isolated from mammalian cells or tissues.Isolated mammalian cell membranes are produced by methods well known inthe art. One example of such a membrane preparation is a microsomalfraction purified from disrupted cells or a tissue sample bydiscontinuous sucrose gradient centrifugation.

Patches with seal resistance of <3 GΩ and access resistance of >50 MΩare discarded. Macroscopic membrane currents are measured during steppulses (600 ms) or during ramp pulses (−140 to +50 mV at 0.32 mV/ms)from a holding potential of −67 mV.

Recording Solutions

For whole cell macroscopic recordings, a nystatin perforated patchtechnique is used, with a bath solution containing (mM): NaCl 130, KCl10, CaCl₂ 1, MgCl₂ 1, HEPES 32.5, glucose 12.5, pH 7.4. The pipettesolution contains (mM): KCl 55, K₂SO4 75, MgCl₂ 8, and HEPES 10, pH 7.2.Nystatin, 50 mg (Calbiochem) is dissolved in dimethylsulfoxide (DMSO), 1ml. Working solutions are made before the experiment by adding 16.5 μlnystatin stock solution to 5 ml of the base pipette solution to yield afinal concentration of nystatin of 165 μg/ml and DMSO 3.3 μl/ml. Thiscomposition of the pipette solution includes K₂SO₄ instead of a portionof the KCl that would otherwise be included. The SO₄ ²⁻ anion, unlikeCl−, is not permeable through the nystatin pore. Reducing the pipette[Cl⁻] reduces the driving force for Cl− into the cell, therebyminimizing osmotic swelling of the cell that might otherwise occurduring electrophysiological recording (Horn and Marty, 1988).

For cell-attached patch recording, a bath solution is used containing(mM): NaCl 130, KCl 10, CaCl₂ 1, MgCl₂ 1, HEPES 32.5, glucose 12.5, pH7.4. The pipette contains (mM): KCl 145, MgCl₂ 1, CaCl₂ 0.2, EGTA 5,HEPES 10, pH 7.28. The measured osmolarity of the extracellular solutionis ≈300 mOsm (Precision Systems, Natick, Mass.).

For most inside-out patch recording, a bath solution is used containing(mM): CsCl 145, CaCl₂ 4.5, MgCl₂ 1, EGTA 5, HEPES 32.5, glucose 12.5, pH7.4. The pipette contains (a): CsCl 145, MgCl₂ 1, CaCl₂ 0.2, EGTA 5,HEPES 10, pH 7.28. For other inside-out patch recordings, Cs⁺ in theabove solutions is replaced with equimolar K⁺.

For the inorganic cation substitution experiments, Cs⁺ in the pipette istypically replaced by equimolar concentrations of individual test ions(Cook et al., 1990).

For outside-out patch recording, the pipette solution contains (mM):CsCl 145, MgCl₂ 1, CaCl₂ 0.2, EGTA 5, HEPES 10, pH 7.28. The standardbath solution contains (mM): CsCl 145, CaCl 24.5, MgCl₂ 1, EGTA 5, HEPES32.5, glucose 12.5, pH 7.4. For the organic cation substitutionexperiments, Cs+ in the bath is replaced with equimolar concentrationsof test cation.

For experiments requiring low concentration of free Ca²⁺ in bathsolution, Ca²⁺-EGTA buffered solution is employed, and free [Ca²⁺] iscalculated using the program WEBMAXC v2.10 available through theStanford University World Wide Web Site. For [Ca²⁺]=1 μM, 5 mM EGTA isused and 4.5 mM Ca²⁺ salt. [Ca²⁺]=1 μM is also used in solutions to testintracellular ATP and Mg²⁺ activities.

Single-channel amplitudes used to calculate slope conductance areobtained by fitting a Gaussian function to an all-points amplitudehistogram of records obtained at various potentials. To calculate openchannel probability (n·Po) at various potentials and with different testagents, the all-points histogram is fit to a Gaussian function and thearea under the fitted curve for the open channel is divided by the areaunder the fitted curve for the closed plus open channel. Values of n·Poat different concentration of test agents are fit to a standard logisticequation using a least-squares method.

For estimating ionic permeabilities of various cations relative to thatfor K⁺, each permeability (Px/PK) is obtained from its reversalpotential (Erev) by fitting to the Goldman-Hodgkin-Katz (GHK) equationwell known in the art. See Goldman 1943; Hodgkin and Katz, 1949.Current-voltage data are fit to the GHK equation, assuming that both K+and the test ion are permeant.

To estimate the pore size of the NC_(Ca-ATP) channel of the presentinvention, the relative permeabilities of organic cations are evaluated.The Stokes-Einstein radius (rse) is calculated from the limitingconductivities (·) of the ions with the formula: r_(SE)·λ=constant. Theconstant is determined from the behavior of TEA at 25° C., for whichλ=44.9 cm²Ω-1, r_(SE)=0.204 nm. The Stoke-Einstein radius is thenconverted to the molecular radius using correction factors read off fromFIG. 6.1 in Robinson and Stokes, 1970. The equivalent limitingconductance for ethanolamine is given (ibid.) and those of other ionsare calculated from their molecular weight by the formula, MW0.5·λ=constant. The constant is determined by the value for ethanolamineat 25° C.: MW=62.1 and λ=4.42 cm^(2Ω-1) equi. Relative permeabilities(Px/PCs) are then plotted against the calculated ionic radii. The effectof solute size on the rate of penetration (permeability) through poresis expressed by the Renkin equation (Renkin, 1955):

a/a ₀=[1−(r/R)]^(2·)[1−2.104(r/R)+2.09(rR)³−0.95(r/R)⁵]  (1)

in which a, a₀, r, and R are the effective area of the pore, the totalcross sectional area of the pore, radius of the solute, and radius ofthe pore, respectively.

Junction potentials are determined with an electrometer by measuring thediffusion potential established across a dialysis membrane and aresubtracted when appropriate. Holding currents are not subtracted fromany of the recordings. Difference currents are obtained by simplysubtracting current records before and after perfusing NaN₃, with noother processing being employed.

Example 1 Morphological Changes with ATP Depletion Using NaN₃

Cultured neural cells have been shown to swell upon ATP depletion. See,Jurkowitz-Alexander et al., 1992; Jurkowitz-Alexander et al., 1993.Freshly isolated NRAs depleted of ATP also results in cell swelling.Ischemia or traumatic injury in brain also causes depletion of ATP inbrain neural cells.

The surfaces of freshly isolated NRAs are highly complex, exhibitingsmall membrane evaginations and fine processes that decorate the entirecell surface, as shown in the scanning electron micrograph in FIG. 14A.Exposure of NRAs to NaN₃ (1 mM) causes changes in the surfaceappearance, characterized early-on by loss of complex structure anddevelopment of surface blebs (FIG. 14B), followed later by a grosslyswollen appearance with complete loss of fine structure and formation ofmultiple large blebs (FIG. 14C). Therefore, NRAs undergo blebbing andswelling after NaN₃-induced ATP depletion.

Phase contrast microscopy is also useful for assessing this process,although fine structure cannot be resolved. Blebbing is visibly apparent10-15 minutes after exposure to NaN₃. Morphological changes of this sortare attributable to loss of cytoskeletal integrity, combined with actionof an osmotic force that causes swelling of the cell.

To assess the contribution of the osmotic gradient to cell swelling, theexperiment is repeated in the presence of mannitol, an impermeantoncotic agent. Mannitol (50 mM), at a concentration sufficient toincrease osmolarity of the extracellular solution from 300 to 350 mOsm,delays bleb formation >30 minutes after exposure to NaN₃. Cellular ATPalso can be depleted using exposure to NaCN (2.5 mM) plus 2-deoxyglucose(10 mM). See, Johnson et al., 1994. Similar morphologic changes,including cell membrane blebbing and delay of blebbing by mannitol areobtained following exposure to NaCN and 2-deoxyglucose. Thisdemonstrates that the effect of NaN₃ is due in fact to ATP depletion andnot to any other non-specific effect of NaN₃.

Example 2 General Electrophysiological Properties of NRAs

The macroscopic currents of whole cell preparations of N u s arecharacterized by small inward currents at negative potentials, largeoutward currents at positive potentials, and a flat “plateau” region atintermediate potentials. NRAs exhibit macroscopic currents that areconsistent with observations in primary cultured cells of the sameorigin. See, Chen et al., 2003; Chen et al., 2001. The NRAs exhibitedinward currents negative to the K⁺ equilibrium potential (E_(K)) areusually <100 pA, much smaller than values reported in cultured neonatalastrocytes (Ransom and Sontheimer, 1995), but consistent with findingsin astrocytes freshly isolated from injured brain (Bordey andSontheimer, 1998; Schroder et al., 1999). The large outward currents inNRAs are partially blocked by charybdotoxin (100 nM), iberiotoxin (100nM) and tetraethylammonium chloride (5 mM), consistent with the presenceof a large conductance Ca²⁺-activated K⁺ channel. See, Perillan et al.,1999. The outward current that remains in the presence of charybdotoxincan be further blocked by 4-aminopyridine (5 mM), and exhibits kineticproperties typical of a delayed rectifier K⁺ channel. Consistent with aprevious report (Perillan et al., 1999), fast inward voltage dependentcurrents attributable to Na⁺ channels are observed in less that 1% ofNRAS.

NaN₃ Elicits Polarizing Inward Current Due to 35 DS Channel

Current clamp recordings are used to investigate the effect of ATPdepletion by NaN₃ in NRAs. For these experiments, a nystatin-perforatedpatch method is used to assure that the metabolic disruption comes fromdrug application and not cell dialysis. Extracellular application ofNaN₃ (1 mM; room temperature) results in a large and swiftdepolarization of the cells (FIG. 1A). NaN₃ rapidly depolarizes thecells to E_(m)≈0 mV (−4.3±0.9 mV). Depolarization usually starts ˜1minute after addition of NaN₃, is complete in <3 minutes, and isirreversible on washout of drug. Ouabain is a known Na⁺/K⁺-ATPaseblocker. See, Brismar and Collins, 1993. The magnitude of thedepolarization observed with NaN₃ far exceeds the small reversibledepolarization induced by ouabain (1 mM). This indicates that the largedepolarization observed after exposure to NaN₃ is not caused byNa⁺/K⁺-ATPase pump failure.

The time course of depolarization with NaN₃ is appreciably more rapidthan the time course for development of cell membrane blebbing observedwith the same treatment. Also, neither the time course nor the magnitudeof the depolarization is affected by raising the extracellularosmolarity with 50 mM mannitol, a treatment that substantially delaysbleb formation. Thus, depolarization is a primary event, not secondaryto cell swelling or stretch.

Voltage-clamp recordings show that exposure to NaN₃ results in a netincrease of inward current in NRAs. Recordings obtained using both ramp(FIG. 1B) and step pulses (FIG. 1C) show significantly larger currentsafter NaN₃ treatment, as shown by comparing the recordings before (a)and after (b) NaN₃ treatment. A plot of the “difference currents”,obtained by subtracting the current-voltage curve before drug from thatafter drug (line c in FIG. 1B), indicates that the new current turned onby NaN₃ reverses near 0 mV. A reversal potential near 0 mV is indicativethat the NaN₃-induced current results from a non-specific cationconductance.

To further characterize the NaN₃-induced current, cell-attached patchrecordings are used. Exposure to NaN₃ elicits single channel currents inpatches that exhibit no single channel currents prior to addition ofdrug (FIG. 1D). After addition of NaN₃, recordings at low temporalresolution reveal a large increase in current variance that, afterincreasing temporal resolution, is revealed to be due to single channelevents (FIG. 1E at 3 and 4). The amplitudes of single-channel eventsrecorded at different membrane potentials are plotted in FIG. 1F, whichshows that NaN₃ activates a single channel conductance of ≈35 pS thatexhibits weak inward rectification when measured in the cell-attachedconfiguration.

Additional experiments are carried out in the cell-attachedconfiguration with the pipette solution supplemented with various drugs.The NaN₃-induced single channel currents are not blocked by 10 mM TEA, 5mM 4-AP, 100 nM iberiotoxin, 100 nM charybdotoxin, or 1 μM tetrodotoxin(4-6 patches for each compound). These experiments indicating that atypical K⁺ or Na⁺ channel is not involved. Also, because 0.2 mM Ca²⁺ isincluded in the pipette solution, these single channel openings areunlikely to be due to monovalent cation influx via an L-type Ca²⁺channel.

Similar depolarization and activation of a 35 pS channel are obtainedwhen cellular ATP is depleted using exposure to NaCN (2.5 mM) plus2-deoxyglucose (10 mM). This demonstrates that the effect of NaN₃ iscaused by ATP depletion and not by any other non-specific effect ofNaN₃.

Apart from ATP depletion, patch excision is also a highly reliablemethod for channel activation. Of the more than 120 cells studied in thecell-attached configuration, spontaneous channel activity attributableto a ≈35 pS conductance is detected in only 2 cells. Thus, theNC_(Ca-ATP) channel of the present invention is typically silent inmetabolically healthy cells. By contrast, a ≈35-pS channel is presentin >90% of inside-out patches formed from NRAs not exposed to NaN₃ orother metabolic toxins, thus demonstrating that an intracellular elementlost on patch excision normally prevents channel activation.

Another potential mechanism of channel activation other than patchexcision is regulatory volume decrease (RVD). Cell swelling is widelyrecognized as a stimulus that initiates RVD, a phenomenon accompanied byactivation of various currents, including a non-selective cation channelin some systems. See, Ono et al., 1994. When membrane patches arestudied in a cell-attached configuration, hyposmotic stimulation (210mosmo/kgH₂O) activated single channel events, but none exhibit a ≈35 pSconductance. This finding indicates that the depolarization and channelactivation observed with NaN³ are not part of an RVD response secondaryto NaN₃-induced cell swelling, and accords with the previously notedobservation that NaN₃-induced depolarization preceded cell swelling.This fact is supported by the observation that the NC_(Ca-ATP) channelis seldom observed in cell attached patches from healthy cells, butbecomes evident in >90% of patches after conversion to an inside-outconfiguration. Also, the NC_(Ca-ATP) channel is lost shortly afterculturing reactive astrocytes.

Example 3 Relative Permeabilities and Pore-Size

The channel is further characterized using membrane patches in theinside-out configuration. Records obtained during test pulses to variouspotentials with equal [K⁺] on both sides of the membrane are shown inFIG. 2A. Amplitude histograms are constructed of events observed atpotentials from −140 mV to +100 mV, and values (mean±SE) for 4 patchesare plotted and show in FIG. 2B. Fit of the data to a linear equationindicates a slope conductance of 35 pS, with an extrapolated reversalpotential (E_(rev)) of +0.1 mV, close to the expected K⁺ reversalpotential (E_(K)) of 0 mV.

In addition to conducting K⁺, the channel transports a variety ofalkaline ions (FIG. 3A), indicating that it is a non-selective cationchannel. In inside-out patches, the conductance of the channel ismeasured with various alkaline ions in the pipette solution, includingCs⁺, Na⁺, Rb⁺, K⁺, and Li⁺, always with equimolar K⁺ in the bathsolution. Current-voltage data are fit to the GHK equation. Na⁺ is shownto have a nearly equal slope conductance (32.6 pS) compared to K⁺ (35.2pS), but the slope conductance is reduced with other cations (FIG. 3B).Measurements of E_(rev) are used to estimate relative permeabilities forthe series of alkaline ions. Values for relative permeabilities derivedfrom the GHK equation are P_(Cs) ⁺/P_(K) ⁺=1.06, P_(Na) ⁺/P_(K) ⁺=1.04,P_(Rb) ⁺/P_(K) ⁺=1.02, and P_(Li) ⁺/PK⁺=0.96, indicating that thischannel is nearly equally permeable to all monovalent cations.

The permeability of the NC_(Ca-ATP) channel of the present invention toanions, such as Cl⁻ is also assessed. After measuring single channelcurrent amplitudes at different potentials with 145 mM KCl, the bathsolution is changed to equimolar K⁺ gluconate. When an agar bridge isused, the solution change resulted in a change in E_(rev)<0.5 mV,indicating that the NC_(Ca-ATP) channel of the present invention isessentially impermeable to anions.

The permeability of the instant channel to divalent cations, Ca²⁺ andMg²⁺, is also investigated (FIG. 3C). When potassium ion in the pipettesolution is replaced with 75 mM Ca²⁺ or Mg²⁺, inward currents are notdetected. Fit to the GHK equation gives best fit values for E_(rev),<<−65 mV for Ca²⁺ and Mg²⁺ respectively, giving relative permeabilitieswith respect to K⁺ of <<0.001, indicating that this channel isessentially impermeable to divalent cations.

Because the NC_(Ca-ATP) channel of the present invention discriminatesvery poorly among monovalent inorganic cations (FIGS. 3A and B),experiments are performed to determine the equivalent pore size of thechannel by measuring channel permeability, relative to Cs⁺, for a widerange of organic cations. Using an outside-out patch configuration,single-channel current-voltage relations are plotted to obtain E_(rev)for a number of organic cations. Permeability ratios are then derivedfrom fits to the GHK equation. For each of the organic cations (a)nethanolamine, (b) guanidium, (c) ethanolamine, (d) diethylamine, (e)piperazine, (f) Tris, and (g) N-methylglucamine, the mean value ofrelative permeability measured is plotted against its hydrated molecularradius (FIG. 3D, empty circles). The permeability ratios define asmoothly declining series of values that are well fit by the Renkinequation. The Renkin equation describes the permeation of a rigid spherethrough a cylindrical pore. Renkin, 1955. Least-squares, fit to theequation, indicates an equivalent pore radius of 0.67 nm for theNC_(Ca-ATP) channel of the present invention. A 0.67 nm pore radius issimilar to pore sizes of 6 A, found for the Ca²⁺ channel (McCleskey andAlmers, 1985) and 7.4 A, found for the nAChR channel (Adams et al.,1980). Junction potentials determined according to the methods describedherein generally did not exceed 5 mV.

Example 4 Inhibition by [ATP]_(i)

The NC_(Ca-ATP) channel is inhibited by intracellular ATP, based on thefinding that this channel is turned on after depleting intracellular ATPby exposure to NaN₃ (See FIGS. 1B, 1C, 1D and 1E) or to NaCN plus2-deoxyglucose. This fact is supported by the observation that theNC_(Ca-ATP) channel of the present invention is seldom observed incell-attached patches from healthy cells, but becomes evident in >90% ofpatches after conversion to an inside-out configuration.

Inside-out patches are used to demonstrate that the channel is sensitiveto block by ATP on the cytoplasmic side of the membrane. Patches arestudied using Cs⁺ as the charge carrier, to assure that no K⁺ channel,such as Kir2.3 or K_(ATP), is contributing to patch activity. With noATP and 1 μM Ca²⁺ in the bath, the NC_(Ca-ATP) channel exhibits vigorousopenings. 1 mM ATP causes profound diminution in channel activity, aneffect that is readily reversed on washout (FIG. 4A); however, channelavailability is unaffected by 1 mM AMP or ADP. The open channelprobability (n·Po) is measured at different [ATP]_(i), and these valuesare normalized to that obtained at [ATP]_(i)=0 mM, and fitted to astandard logistic equation. As shown in FIG. 4B, the NC_(Ca-ATP) channelis blocked by [ATP]_(i) in a dose-dependent manner. Half maximuminhibition (IC₅₀) is observed at [ATP]_(i), =0.79 μM with a Hillcoefficient of 1, and channel activity is completely abolished at[ATP]i>30 μM. ADP and AMP, have no effect on the NC_(Ca-ATP) channelactivity in inside-out patches.

This in vitro assay for determining the concentration of the testcompound which achieves a half-maximal inhibition of channel activity(ICSO) may be used to formulate dose in animal models to achieve acirculating plasma concentration range that includes the IC₅₀.

Example 5 Activation by [Ca²⁺]_(i)

The Ca²⁺ concentration on the cytoplasmic side of the membrane is alsofound to regulate activity of the NC_(Ca-ATP) channel of the presentinvention. The relationship between NC_(Ca-ATP) ATP channel activity and[Ca²⁺]_(i) is examined using inside-out patches studied at membranepotential (Em)=−80 mV. Changing [Ca²⁺]_(i) clearly affects activity ofthe NC_(Ca-ATP) channel (FIG. 5A). When free [Ca²⁺]_(i) is <30 nM, nochannel activity is apparent. With [Ca²⁺]_(i)>30 nM, the openprobability (n·Po) increases in accordance with the [Ca²⁺]_(i), up to ≈1μM of [Ca²⁺]_(i) at which activity is near maximum. The effect of Ca²⁺on channel availability is found to depend on membrane voltage. Valuesof nP·o from 4-9 patches obtained at three different potentials, Em=−40mV, −80 mV and −120 mV, are normalized to values observed with 3 μM[Ca²⁺]_(i). These data are fit to a standard logistic equation using aHill coefficient of 1.5 and half-maximum values of 0.12 μM, 0.31 μM and1.5 μM at −40 mV, −80 mV and −120 mV, respectively (FIG. 5B). These dataindicate that channel activity is strongly dependent on [Ca²⁺]_(i) atphysiologically relevant concentrations, and that the effect of Ca²⁺ isvoltage dependent, consistent with a Ca²⁺ binding site inside theelectric field of the membrane.

Example 6 Internal Mg²⁺ Causes Rectification

Because certain channels are sensitive to intracellular Mg²⁺ (Chuang etal., 1997; Perillan et al., 2000), experiments are carried out todetermine whether the channel rectification observed in cell-attachedpatch recordings (see FIG. 1F) might be due to intracellular Mg²⁺. Usinginside-out patches studied with equimolar K⁺ on both sides of themembrane, [Mg²⁺] is varied on the cytoplasmic side. Single channelrecords and channel amplitudes observed with different [Mg²⁺]_(i) areshown (FIG. 6). No rectification is evident with [Mg²⁺]_(i) 30 μM, butat [Mg²⁺]_(i)≈100 μM, increasingly strong rectification is present. At100 μM, Mg²⁺ appears to produce a flickery block.

Example 7 Identifying the Presence of SUR in NRAs

To determine if SUR receptors are present in NRAs, the binding ofglibenclamide to these cells is assessed by fluorescence microscopy.Eight week old Wistar rats are injured by a stab wound into thesubcortical white matter and implantation of a gelatin sponge aspreviously described herein. Eight days later, tissue sections offormaldehyde-fixed brains from injured animals are incubated for 60minutes at room temperature with 20 nM FITC-conjugated glibenclamide. Afluorescence image of the gelatin sponge shows labeled cells lining thecavities of the sponge. In brain adjacent to the injury, essentially noglibenclamide binding is apparent. These data indicate that SUR, whichare not normally present in subcortical white matter, are expressed inneural cells following traumatic injury.

RT-PCR

Total RNA is extracted from cells and used to synthesize cDNA, which isamplified from reactive astrocytes is analyzed by RT-PCR on an agarosegel stained with ethidium bromide. FIG. 7A is a photograph of the gelshowing the RT-PCR for SUR1 and SUR2. FIG. 7B is a photograph of a gelshowing the RT-PCR for Kir6.1 and Kir6.2. Lanes 3 and 4 in FIGS. 7A and7B show the RT-PCR for insulinoma cells. Lanes 5 and 6 show the RT-PCRfor reactive astrocytes. Lane 1 in FIGS. 7A and 7B represents laddersize markers; Lane 2 in FIGS. 7A and 7B is a blank control. In FIG. 7A,lanes 3 and 4 show the SUR1 and SUR2 experiments, respectively, ininsulinoma cells. Insulinoma cells are known to express SUR1, but notSUR2. Lanes 5 and 6 in FIG. 7A show the SUR1 and SUR2 experiments inreactive astrocytes, respectively. FIG. 7A shows that SUR1 mRNA ispresent in reactive astrocytes, as well as in the control insulinomacells. SUR2 is absent in both cell types. In FIG. 7B, lanes 3 and 4 showthe Kir6.1 and Kir6.2 experiments in insulinoma cells, respectively.Kir6.1 is present in insulinoma cells, but Kir6.2 is not. Kir6 is thepotassium channel associated with SUR1 in insulinoma cells. Lane 5 and 6in FIG. 7B show that neither Kir6.1 nor Kir6.2 is present in reactiveastrocytes. Therefore, reactive astrocytes express SUR1 mRNA, but Kir6.1and Kir6.2 mRNA is absent from the cells.

The presence of SUR1 in reactive astrocytes combined with the regulationof the NC_(Ca-ATP) channel in astrocytes by SUR antagonists indicatesthat SUR regulates the NC_(Ca-ATP) channel of the present invention.

Example 8 Tryptic Digests

A characteristic feature of SUR-regulated K_(ATP) function is thattryptic digestion of the cytoplasmic face of the channel, but not itsextracellular face causes loss of inhibition by sulfonylureas, withoutaltering sensitivity to ATP and without changing the biophysicalproperties of the channel. The effect of trypsin on NC_(Ca-ATP) functionis shown in FIG. 8. Under control conditions, channel activity in theinside-out patch configuration is strongly inhibited by 1 μMglibenclamide. Exposure to 100 pg/ml trypsin on the cytoplasmic side ofthe membrane for 3 minutes yields a patch that still exhibits strongchannel activity, but that channel activity is completely unaffected byglibenclamide. After such trypsin treatment of the cytoplasmic side, thebiophysical properties of the channel, including open channelconductance, open channel times, Ca²⁺-mediated activation are unchanged,and the channel still maintains its typical sensitivity to ATP. Bycontrast, exposure of the extracellular side of the membrane has noeffect on glibenclamide inhibition. These trypsin digest data on theNC_(Ca-ATP) channel of the present invention provide additionalsupporting evidence that SUR1 is involved in regulation of theNC_(Ca-ATP) channel, because the results compare to previous findingsfrom SUR1-regulated K_(ATP) channels. Linkage of a SUR to anon-selective ATP sensitive cation channel, has not been shownpreviously.

Assays for Compounds or Compositions that Block NC_(Ca-ATP) Channel andInhibit Neural Cell Swelling Example 9 Effects of Sulfonylurea Compounds

Sulfonylurea compounds are known to modulate the sulfonylurea receptor.A sulfonylurea receptor is generally associated with K_(ATP) channels asa regulatory component, and is found in various tissues, including ratNRAs. Notably, the K_(ATP) channels Kir6.1 and Kir6.2 are not present inrat NRAs (FIG. 7B). It is possible to activate the NC_(Ca-ATP) channelwith SUR ligand diazoxide in outside-out patches (FIGS. 9A and 9B). NaN₃does not elicit channel activity in isolated membrane patches,indicating that it works via ATP depletion rather than any direct effecton the channel.

Example 10 In Vitro Assays for Determining Dose-Dependent Blockage ofthe NC_(ca-Atp) Channel

SUR1 blocking compounds, such as glibenclamide and tolbutamide, areknown to have an inhibitory effect on K_(ATP) channels. In oneembodiment, the present invention arrives at the objects of theinvention by providing a method in which the direct inhibitory effect ofglibenclamide and tolbutamide on NC_(Ca-ATP) channels is determined(FIGS. 10 and 11). Inside-out patches are used to show the inhibitoryeffect of sulfonylureas. To ensure that no K⁺ channel, particularlyK_(ATP) is contributing to patch current, Cs⁺ is used as the chargecarrier. Channel activity is profoundly diminished by the addition of 10μM glibenclamide (FIG. 10A at b), and the activity is shown to be due toa 35 pS cation channel, which is consistent with the NC_(Ca-ATP) channelof the present invention (FIG. 10C). Another sulfonylurea, tolbutamide,is also shown to inhibit NC_(Ca-ATP) channel activity (FIGS. 11A and11B). As shown in FIG. 11B, the NC_(Ca-ATP) channel is blocked by thesulfonylureas in a dose-dependent manner. With tolbutamide, half maximuminhibition (EC₅₀) is observed at 16.1 μM with a Hill coefficient of 1.3,and channel activity is completely lost at concentrations >300 μM. Withglibenclamide, EC₅₀ is observed at 48 μM with a Hill coefficient of 1.2.The sensitivity of the NC_(Ca-ATP) channel of the present invention toblocking in NRAs with both of these sulfonylurea compounds correspondsclosely to that reported in pancreatic β cells and in expression systemswith SUR1, but not SUR2.

This in vitro assay for determining the concentration of the testcompound which achieves a half-maximal inhibition of channel activitymay be used to formulate dose in animal models to achieve. a circulatingplasma concentration range.

Example 11 Mechanism of Channel Regulation by Sulfonylureas

The NC_(Ca-ATP) channel of the present invention exhibits two openstates, with a shorter and a longer dwell time, each less than 10 ms.FIG. 12 shows data from a patch exhibiting an open channel probability(n·Po) of 0.63, with open dwell time values τ₀₋₁ τ₀₋₂ and of 1.9 and 8.2ms. After successive application of 3 μM tolbutamide (FIGS. 12B and 12E)and 30 μM tolbutamide (FIGS. 12C and 12F), n·Po decreased to 0.44 and0.09, respectively, but the open dwell time values are not appreciablyaffected by the drug. Closed channel dwell times are increased induration and frequency by tolbutamide (FIGS. 12H and 12I). Thus, thechannel of the present inventions exhibits a form of channel inhibitionin which the blocking compound had no effect on open channel dwell timesand a progressive increase in long closures. This form of channelinhibition is similar to that produced by sulfonylureas acting on theK_(ATP) channel in pancreatic β cells. See, Gillis et al., 1989;Babeenko et al., 1999).

Example 12

Application of 100 μM of the SUR-activator diazoxide activates the 35 pSchannel of the present invention, causing weak inward rectification incell-attached patches (FIGS. 13A, 13B and 13C). To determine the type ofSUR affecting activation of the NC_(Ca-ATP) channel of the presentinvention, experiments are conducted using sulfonylurea compounds thatpreferentially activate SUR2 over SUR1, namely cromakalin, andpinacidil. Both cromakalin and pinacidil had no effect on theNC_(Ca-ATP) channel of the present invention, which is consistent withother data described herein indicating that SUR1 is associated with theNC_(Ca-ATP) channel of the present invention, and activation of thechannel is not mediated by SUR2.

Example 13 Sur-Mediated Cell Swelling

After addition of NaN₃ to deplete ATP in cells, cell blebbing typicallybecomes apparent in 7-10 minutes. Diazoxide is an SUR1 agonist or SUR1activator. When diazoxide alone is added to the cells, blebbing occurseven without ATP depletion, Diazoxide, therefore, opens the channeldirectly without ATP depletion by activating SUR1. However, when cellsare pretreated with glibenclamide, addition of NaN₃ does not causeblebbing, even after 30 minutes. Thus, activation of NC_(Ca-ATP) channelby ATP depletion or by the channel opener, diazoxide, can result inblebbing and swelling of NRAs, and that swelling can be prevented byblocking the channel with glibenclamide. ATP depletion by Na azide canresult in necrotic cell death of NRAs. These findings accord with thedata described herein that glibenclamide protects from the opening ofthe NC_(Ca-ATP) channel following ATP depletion, and that opening ofthis channel is responsible for cell blebbing.

The antagonist used in the methods of the present invention includes acompound that interferes with NC_(Ca-ATP) function. Typically, theeffect of an antagonist is observed as a blocking of NC_(Ca-ATP) currentin conditions under which the channel has been activated and current canbe measured in the absence of the antagonist.

In addition to SUR1 specific sulfonylurea compounds, agents that blockSUR1, also include compounds that are structurally unrelated tosulfonylureas. Such SUR1 blockers include a class of insulinsecretagogues compounds that bind to the SUR, which were identified anddeveloped for the treatment of type 2 diabetes. The benzarnidoderivatives: repaglinide, nateglinide, and meglitinide represent onesuch class of insulin secretagogues, that bind to the SUR. Nateglinideis an amino acid derivative. Also, imidazoline derivatives have beenidentified that interact with the sulfonylurea receptor (SUR) 1 subunitsuch as midaglizole (KAD-1229), LY397364 and LY389382.

In one preferred embodiment of the present invention, compounds thatpreferentially block SUR1, but not SUR2, are used in the method of thepresent invention. Such compounds include tolbutamide and gliclazide.The following compounds block both SUR1 and SUR2: glibenclamide,glimepiride, repaglinide, and meglitinide. In yet another embodiment ofthe method of the present invention, administration is combined withMgADP, which has been show to produce an apparent increase ofsulfonylurea efficacy on channels containing SUR1, but not SUR2.

Example 14

To determine whether NC_(Ca-ATP) activation by ATP depletion initiatesnecrosis of reactive astrocytes that express this channel, studies areconducted to determine if glibenclamide is capable of protectingreactive astrocytes from cell death by inhibiting NC_(Ca-ATP) channelactivity via its action on SUR1. Two types of cell death, apoptosis andnecrosis, are assessed following ATP depletion.

Thus, activation of NC_(Ca-ATP) channel is responsible for necroticdeath of NRAs following ATP depletion, and that glibenclamide canprevent this form of cell death.

In this Example, the preparation of freshly isolated NRAs was furtherpurified by removal of RBCs, as described herein to provide a cellpopulation having <1% RBCs. Over 95% of cells had resting potentialsnear E_(K), suggesting that the enzymatic dissociation method had notappreciably harmed the cells. Over 95% of cells are positive for theastrocyte marker, glial fibrillary acidic protein (GFAP) as determinedby immunofluorescence. When examined by phase microscopy, the NRAs areof various sizes, ranging from 11-45 μm in diameter, some of which arephase bright and others are phase dark. A subgroup of phase bright cellshad multiple short but distinct cell processes that are shorter than thecell soma. In this Example, only larger (≈30 μm diameter), phase brightcells with short processes (<1 cell length) are studied. This populationof NRAs reliably express NC_(Ca-ATP) channels.

Experiments are conducted at room temperature (22-25° C.) within 24 hrof cell isolation. An aliquot of cells is placed on a chamber slide(LAB-TEK, Naperville, Ill.) filled with extracellular bath solutioncontaining (a): NaCl 130, KCl 10, CaCl₂ 1, MgCl₂ 1, HEPES 32.5, glucose12.5, pH 7.4. After viable cells adhered to the surface, any residualdebris not previously removed by centrifugation is washed away byflushing with excess solution. Cells are subjected to ATP depletion by 1mM Na azide to activate (open) the NC_(Ca-ATP) channels, and thenincubated with glibenclamide (1 μM).

Thereafter, the cells are examined by propidium iodide (PI) staining forevidence of cellular membrane permeabilization, an indication of earlyoncotic or necrotic cell death. See, Barros et al., 2001. The cells arealso examined by fluorescein-tagged annexin V binding for evidence ofexternalization of the phosphoaminolipid phosphotidylserine from theinner face of the plasma membrane to the outer surface, an earlyindication of apoptosis. See, Clodi et al., 2000; Rucker-Martin et al.,1999. Staining procedure are conducting according to manufacturedirections (Vybrant Apoptosis Assay Kit 2, Molecular Probes). Slides aremounted using ProLong antifade mounting medium (Molecular Probes).Signals are visualized using a Nikon Diaphot epifluorescent microscope(Leitz Wetzlar). Images are captured and stored using a SenSys digitalcamera (Roper Scientific Inc.) and IPLab software (version 3.0;Scanalytics Inc.). Annexin V-positive cells or PI-positive cells arecounted in 20 individual fields using a 20× objective lens. Mean valuesof positive cells in 20 fields for various treatment groups are comparedusing ANOVA Pairwise multiple comparisons, with p<0.05 being consideredas indicating a significant difference.

The fluorescence microscopy photos shown in FIG. 15A show that underbaseline (control) conditions, both annexin V-positive and PI-positivecells (photos a and d, respectively) are rare in the cell isolates.After a 10-min incubation with Na azide (1 mM), the number ofPI-positive cells increased substantially (p<0.05) (FIG. 15A at photo band FIG. 15B). This indicates that ATP depletion triggers necrotic deathin these cells. By contrast, Na azide treatment caused the number ofannexin V-positive cells to increase slightly; the increase not beingstatically significant (p>0.05) (FIG. 15A at photo e and FIG. 15C). Thisindicates that apoptotic death was not a major endpoint of ATP depletionin these cells.

Pretreatment of cells with glibenclamide (1 μM) at the time ofadministration of Na aide dramatically decreased the number ofPI-positive cells (p<0.05; FIG. 15A at photo c and FIG. 15B), indicatingsignificant protection from necrotic death following ATP depletion. Thenumber of NRAs undergoing apoptotic death also decreased withglibenclamide, as indicated by annexin V labeling (FIG. 15A at photo fand FIG. 15C), but values for this group were not significantlydifferent.

This data indicate that the NC_(Ca-ATP) channel is involved in themechanism of the necrotic cell death of reactive astrocytes. ThisExample shows that necrotic, rather than apoptotic, cell death is theprincipal endpoint of ATP depletion in these cells. Therefore, ATPdepletion by Na azide initiates cell death by removal of the ATP blockof the NC_(Ca-ATP) channel, thus initiating oncotic cell swelling.Involvement of this channel in oncotic cell swelling is confirmed byshowing that necrotic death can also be induced by diazoxide, thechannel opener that activates the NC_(Ca-ATP) channel in these cells,and could be blocked by glibenclamide, which prevents opening of theNC_(Ca-ATP) channel. The involvement of the NC_(Ca-ATP) channel in celldeath of reactive astrocytes provides a mechanism and target of death inthese cells, as well as the importance of blocking the NC_(Ca-ATP)channel to prevent the death of reactive astrocytes, which occurs intraumatic brain injury.

Example 15 In Vitro Assays for Determining the Ability of a TestCompound to Provide Dose-Dependent Blockage of Nc_(ca-Atp) Channel

NC_(Ca-ATP) channels blocking compounds can be identified by a method inwhich the direct inhibitory effect of the test compound on NC_(Ca-ATP)channels is determined Inside-out patches are used to show theinhibitory effect of the compound. To ensure that no K⁺ channel,particularly K_(ATP) is contributing to patch current, Cs⁺ is used asthe charge carrier. Compounds that profoundly diminish channel activity,and the activity is shown to be due to a 35 pS cation channel, such acompound is identified as a compound that blocks the NC_(Ca-ATP)channels and is capable of inhibiting neuronal cell swelling and brainswelling. Varying concentrations of the compound are used to determinewhether the NC_(Ca-ATP) channel is blocked by the compound in adose-dependent manner. The concentration at which half maximuminhibition (EC₅₀) is observed and the concentration at which channelactivity is completely lost are determined. The sensitivity of theNC_(Ca-ATP) channel of the present invention to blocking in NRAs withthe test compound can be compared. This in vitro assay for determiningthe concentration of the test compound which achieves a half-maximalinhibition of channel activity may be used to formulate dose in animalmodels to achieve a circulating plasma concentration range.

Example 16 In Vivo Assays for Determining Dose-Dependent Blockage of theNc_(ca-Atp) Channel

The concentration of the test compound which achieves a half-maximalinhibition of channel activity is used to formulate dose in animalmodels to achieve a circulating plasma concentration range. The dose oftest compound that achieves a circulating plasma concentration rangecalculated by methods known in the art is administered to an animalhaving brain injury or cerebral ischemia. To determine whether the testcompound prevents, inhibits or diminishes brain swelling, the epiduralpressure and/or intracranial pressure of the animal is measured, such asby using a microballoon, to quantitatively monitor brain swelling. Also,the swelling can be monitored by magnetic resonance (MR) imaging. Threedifferent studies start administration prior to, at the time of, orafter the brain injury. A compound that provided diminishes brainswelling, as compared to controls, is identified as a compound capableof inhibiting neuronal cell swelling and brain swelling. Varyingconcentrations of the compound are used to determine whether thecompound delivers efficacy in a dose-dependent manner. The dose at whichhalf maximum inhibition is observed and the concentration at which brainswelling is most quickly alleviated are determined Formulations areproduced comprising the optimal effective dose of the test compound forpreventing, inhibiting, or diminishing brain swelling, along with apharmaceutically acceptable carrier.

Example 17 Additional Mechanisms for Maintaining NRAs in a PolarizedState

When reactive astrocytes are strongly depolarized due to opening of theNC_(Ca-ATP) channel, they undergo blebbing and swelling and eventuallysustain necrotic cell death. As stated above, when reactive astrocytesare strongly depolarized due to opening of a non-selective channel thatis sensitive to Ca²⁺ and ATP (NC_(Ca-ATP) channel), they undergoblebbing and swelling and eventually sustain necrotic cell death. Thedeath of these reactive astrocytes can be prevented if strongdepolarization can be prevented, in other words, if the cells can bemaintained in a polarized state.

One potential way of maintaining the NRAs in a polarized state is toopen the Kir2.3 channel. NRAs are exposed to the Kir2.3 channel opener,Tenidap(5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H-indole-1-carboxamide),to maintain Kir2.3 channels open. Native reactive astrocytes freshlyharvested from adult rat brains after injury are exposed to Tenidap toevaluate the drug's ability to open the Kir2.3 channel in these cells.Preferably, type 1 reactive (R1) astrocytes are harvested and used inthis assay. One of the subtypes of reactive astrocytes is the type R1astrocyte. Type R1 astrocytes comprise the largest population ofrecoverable astrocytes at the site of brain injury. They arecharacteristically located in the region of tissue surrounding theinjury site, many of which are found to have migrated into the injurysite itself. See, Perillan, et al., 1999.

The reactive astrocytes that are part of the cellular response to TBIand stroke are comprised of at least two subtypes. One of the subtypesof reactive astrocytes is the type R1 astrocyte. Type R1 astrocytescomprise the largest population of recoverable astrocytes at the site ofbrain injury. They are characteristically located in the region oftissue surrounding the injury site, with many of these cells also beingfound to have migrated into the injury site itself. See, Perillan, etal. 1999.

Type R1 astrocytes are the predominant type of reactive astrocyte in theNRA preparations. Type R1 astrocytes express two critically importantion channels in their cell membrane: (a) the Kir2.3 channel, which ispresent in cultured as well as freshly isolated cells; and (b) theNC_(Ca-ATP) channel, which is present only in freshly isolated reactiveastrocytes and lost shortly after culturing. The Kir2.3 is an inwardrectifier channel that is critically important for maintaining the cellpolarized to a normal resting potential near the potassium reversalpotential (≈−75 mV). When this channel is inactivated or inhibited, thecell depolarizes to a potential near the chloride reversal potential(≈−25 mV). Characteristic features of the NC_(Ca-ATP) channel are: 1) itis a non-selective cation channels that allows passage of Na⁺, K⁺, andother monovalent cations quite readily; 2) it is activated by anincrease in intracellular calcium, and/or by a decrease in intracellularATP; and 3) it is regulated by sulfonylurea receptor type 1 (SUR1). SUR1had been considered to be associated exclusively with K_(ATP) channels,such as those found in pancreatic β cells.

Opening of the NC_(Ca-ATP) channel following ATP depletion, as withischemia or hypoxia, causes depolarization of the cell due to influx ofNa⁺. This influx of Na⁺ increases the osmotic load within the cell, andas a result, H₂O enters the cell to equilibrate the osmotic load. Theresult is an excess of Na⁺ and H₂O intracellularly, a pathologicalresponse that produces cell blebbing and cell swelling and that is knownas cytotoxic edema. Left unchecked, this pathological responseeventually leads to cell death. As disclosed herein, this cell death ismostly necrotic cell death but to a lesser extent, apoptotic cell deathas well.

A number of approaches may be used to meliorate brain swelling due tocytotoxic edema. One currently used treatment for treating patients inrelevant clinical situations is based on increasing extracellularosmolarity to reduce the driving force for influx of H₂O. This strategyalso reduces blebbing in isolated cells.

A more specific strategy to reduce cytotoxic edema is inactivating orblocking the NC_(Ca-ATP) channel that is primarily responsible for theinflux of Na⁺ that draws H₂O into the cell and that actually causescytotoxic edema. One highly selective approach to inactivating thischannel is to exploit the unique relationship between the channel andthe controlling regulatory subunit, SUR1. A variety of drugs have beendeveloped that interact with SUR1 in pancreatic β cells to block theK_(ATP) channel in those cells and thereby treat diabetes. Some of thesedrugs belong to the class of agents called sulfonylureas. As describedherein, drugs that block the K_(ATP) channel, such as glibenclamide andtolbutamide, are highly effective at blocking the NC_(Ca-ATP) channel intype R1 astrocytes. Drugs capable NC_(Ca-ATP) channel blocking in NRAs(a) prevents cell blebbing in response to ATP depletion, (b)significantly reduces cell death following ATP depletion. Also, the useof glibenclamide to treat brain swelling in an animal suffering fromstroke or brain injury is described herein.

Yet another strategy to oppose the effect of the NC_(Ca-ATP) channel andreduce cytotoxic edema would be to counteract depolarization of the cellthat accompanies opening of the NC_(Ca-ATP) channel. One way toaccomplish this is to enhance opening of the Kir2.3 channels that arealso present in these cells. An anti-inflammatory compound, Tenidap(5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H-indole-1-carboxamide),is an opener of Kir2.3 channels. See, Popp et al., 1992; Liu et al.,2002. Tenidap is evaluated for its ability to reduce cell blebbing andswelling and necrotic cell death in response to ATP depletion in theisolated cells as well as in situ in injured rat brain. To assesswhether Tenidap opens the Kir2.3 channels in type R1 astrocytes, usingmethods similar to those described herein for evaluating the status ofthe NC_(Ca-ATP) channel. Results from such experiments that show Tenidapto open Kir2.3 channels in type R1 astrocytes, and reduce cell blebbingand cell death in response to ATP depletion would indicate theusefulness of Tenidap in treating brain swelling and cytotoxic edemaresulting from TBI or cerebral ischemia. The effective amount of Tenidapis that amount capable of reducing brain swelling or cerebral ischemiadue to the drug's ability to inhibit neural cell swelling and necroticcell death.

SUR1 blockers are believed to be the most specific, reliable blockersand to provide the fewest untoward side effects. Further, a combinationof treatments including use of two to more of osmotic diuretics,NC_(Ca-ATP) channel blockers such as glibenclamide and Kir2.3 channelopeners such as Tenidap may provide excellent efficacy in amelioratingcytotoxic edema and reducing morbidity and mortality in brain injury andstroke. Thus, comcomitant or successive administration of an NC_(Ca-ATP)channel blocker and a Kir2.3 channel opener is expected to provideexcellent efficacy in ameliorating cytotoxic edema and reducingmorbidity and mortality in brain injury and stroke. For example,administration of glibenclamide and Tenidap would be useful forameliorating cytotoxic edema and reducing morbidity and mortality inbrain injury and stroke.

Example 18 Modulation by Estrogen

A characteristic feature of K_(ATP) channels (Kir6.1, Kir6.2) is thatchannel affinity for ATP is modulated by the presence of the membranelipid, PIP₂. The open-state stability of K_(ATP) channels is increasedby application of PIP₂ to the cytoplasmic side of the membrane(Ashcroft, 1998; Baukrowitz et al., 1998; Rohacs et al., 1999). Anincrease in the open-state stability is manifested as an increase in thechannel open probability in the absence of ATP, and in a correspondingdecrease in sensitivity to inhibition by ATP (Enkvetchakul et al., 2000;Haruna et al., 2000; Koster et al., 1999; and Larsson et al., 2000).

Given the numerous similarities between the K_(ATP) channel and theNC_(Ca-ATP) channel, the inventors postulated that ATP-sensitivity ofthe NC_(Ca-ATP) channel would respond to PIP₂ in the same way. This wastested by studying NC_(Ca-ATP) channels in inside out patches with Cs⁺as the charge carrier, and with 1 μM Ca²⁺ and 10 μM ATP in the bath,with the latter expected to fully block the channel. Under theseconditions, only the NC_(Ca-ATP) channel was recorded in R1 astrocytes.When PIP₂ (50 μM) was added to the bath, channel activity becameprominent (FIG. 16), as predicted by analogy to the effect of PIP₂ onK_(ATP) channels. This channel activity was blocked by glibenclamide,confirming identity of the channel.

To determine if a receptor-mediated mechanism was involved in themodulation of NC_(Ca-ATP) channel activity, a well known phospholipase C(PLC) was used to study if PLC activation would cause degradation andconsumption of PIP₂ and thereby increase affinity for ATP, e.g., reducechannel opening. Estrogen is a well known PLC activator in brain as wellas elsewhere (Beyer et al., 2002; Le Mellay et al., 1999; Qui et al.,2003). For this experiment, cell attached patches were studied toprevent alteration of intracellular signaling machinery. NC_(Ca-ATP)channel activity was produced by exposure to Na azide to cause depletionof cellular ATP (FIG. 2, initial part of the record).

When estrogen (E2; 10 nM) was applied to the bath, activity due to theNC_(Ca-ATP) channel was soon terminated (FIG. 17). This suggested thatestrogen exerted regulatory control over the NC_(Ca-ATP) channel, andsuggested that an estrogen receptor capable of rapid (non-genomic)activation of signaling cascades was present on these cells.

Next, to determine whether estrogen receptors could be detected in R1astrocytes from males and females. Gelatin sponge implants wereharvested 7 days after implantation in a group of 3 female rats (F) andanother group of 3 male rats (M). Pooled protein from each group wasanalyzed at 2 dilutions (4×=50 μg total protein; 1×=12.5 μg totalprotein) by Western blotting, with protein from uterus being used as acontrol (FIG. 18A). Membranes were blotted with an antibody thatrecognized both α and β estrogen receptors. Both males and femalesshowed prominent bands at the appropriate molecular weights for the α(66 kDa) and β (55 kDa) receptors (FIG. 18) (Hiroi et al., 1999). Thesame samples of protein from males and females were also used to confirmpresence of SUR1, with protein from pancreas used as a positive control(FIG. 18B). Notably, estrogen receptors have previously been reported inastrocytes from males and females (Choi et al., 2001). In cerebralcortex, the β isoform is reportedly more abundant (Guo et al., 2001) assuggested by the Western blot.

Next, the electrophysiological experiment of FIG. 17 was repeated usingR1 astrocytes harvested from male rats. As above, cell attached patcheswere studied in which NC_(Ca-ATP) channel activity was activated bydepletion of intracellular ATP following exposure to Na azide (FIG. 4A).Examination of the record at higher temporal resolution confirmedactivity of a well defined channel of the appropriate conductance forthe NC_(Ca-ATP) channel (FIG. 4B). When estrogen was applied to the bath(FIG. 4, E2, 10 nM, arrow), activity due to the NC_(Ca-ATP) channel wasquickly terminated (FIG. 19). These data provided further evidence thatestrogen exerted regulatory control over the NC_(Ca-ATP) channel, andsuggested, in addition, that this response was equally robust in R1astrocytes from males and females.

By analogy to the effects of estrogen, other mechanisms that depletePIP₂, including other receptor-mediated mechanism as well as more directactivators of PLC such as G-proteins etc., would be expected to have asimilar inhibitory effect on activity of the NC_(Ca-ATP) channel andthereby exert a protective effect.

Example 19 The Gliotic Capsule

The standard model involved placing a stab injury into the parietal lobeof an anesthetized rat and implanting a sterile foreign body (gelatinsponge; Gelfoam®) into the stab wound. Variants of the standard modelincluded impregnating the sponge with a substance (e.g.,lipopolysaccharide, LPS) or infusing a substance continuously in vivousing an osmotic mini-pump with the delivery catheter placed directlyinto the sponge. The injury procedure was well tolerated by the animals,with virtually no morbidity or mortality and minimal pain. After anappropriate time in vivo, the whole brain was harvested for histologicalor immunohistochemical study of tissue sections. Alternatively, if thesponge itself was gently removed from the brain, the inner zone of thegliotic capsule adheres to the sponge and was excised along with it.Thus, the sponge was assayed for protein (e.g., Western) or mRNA(RT-PCR), or it was enzymatically dissociated to yield constituent cellsfor electrophysiological or other single-cell measurements.

The gliotic capsule was well developed 7-10 days after injury. Thegliotic capsule was visualized in coronal sections by perfusing theanimal with Evans Blue prior to perfusion-fixation of the brain (FIG.20A). A region of edema (dark) was seen to outline the avascular glioticcapsule (light) that surrounded the gelatin sponge (dark)Immunohistochemical examination with anti-GFAP antibodies showed thatthe brain parenchyma in the vicinity of the sponge harbors manyGFAP-positive reactive astrocytes (FIG. 20B; arrow showed where thegelatin sponge was). At higher power, these intraparenchymalGFAP-positive cells were shown to be large and to bear many prominentcell processes (FIG. 20C, arrow). Examining the gelatin sponge itselfshowed GFAP-positive reactive astrocytes that migrated into theinterstices of the sponge (FIG. 20D, arrow).

Example 20 Isolation of Cells from the Gliotic Capsule

Phase contrast microscopy of cells freshly isolated by papain digestionof the inner zone of the gliotic capsule and gelatin sponge revealedthree types of cells. Most of the cells (>90%) were large, round, haveno cell processes and were phase-bright (FIG. 21A). A number of cells(3-5%) were small, round, have no cell processes and were phase-dark(FIG. 21B). Occasionally, a cell was found that was intermediate insize, was phase-bright and had multiple processes that were more thanone cell diameter in length (Chen et al., 2003) Immunofluorescence studyshowed that all of these cells were strongly positive for typicalastrocyte markers, including GFAP (FIG. 21C,D) and vimentin (FIG.21E,F). Microglia were not prominent in the inner zone of the glioticcapsule itself, as indicated by sparse labeling for OX-42. Cells of theinner zone of the gliotic capsule were negative for the 02A progenitormarker, A2B5, and the fibroblast marker, prolyl 4-hydroxylase (Dalton etal., 2003).

As with freshly isolated cells, three morphologically distinct types ofcells were observed in primary culture. Most cells (>90%) were largepolygonal cells (FIG. 21Gb), a few (3-5%) were small bipolar cells (FIG.21Ga), and only occasionally were process-bearing stellate-shaped cellsobserved (Perillan et al., 2000). All of these cells were stronglylabeled with anti-GFAP antibodies (FIG. 21H). Experiments in which cellsobtained by enzymatic digestion were followed individually in primaryculture showed that the large phase-bright cells develop into largepolygonal cells (FIG. 21Gb), and the small phase-dark cells developedinto small bipolar cells (FIG. 21Ga) (Dalton et al., 2003).

The three morphologically distinguishable types of GFAP-positiveastrocytes from the inner zone of the gliotic capsule exhibited verydifferent macroscopic whole cell electrophysiological profiles:

(i) Electrophysiological studies on stellate astrocytes showed that theyexpressed Kir2.3 and Kir4.1 inward rectifier channels, andimmunolabeling experiments suggested that they also expressed K_(ATP)channels comprised of SUR1 and Kir6.1 subunits (Chen et al., 2003;Perillan et al., 2000);

(ii) Electrophysiological studies on R2 astrocytes showed that theyexpressed a novel Ca²⁺-activated Cl− channel that was sensitive to thepolypeptide toxin from the scorpion, Leiurus quinquestriatus (Dalton etal., 2003). Only the R2 astrocyte expressed this channel.

(iii) Electrophysiological studies on R1 astrocytes showed that theyexpress Kir2.3 inward rectifier channels that are regulated by TGFβ1 viaPKCδ (Perillan et al., 2002; Perillan et al., 2000). When freshlyisolated but not after culturing, R1 astrocytes also expressed a novelSUR1-regulated NC_(Ca-ATP) channel (Chen et al., 2003; Chen et al.,2001).

Example 21 Expression of SUR1

Glibenclamide binds to sulfonylurea receptors, SUR1 and SUR2, withhigher affinity for SUR1. Immunofluorescence studies were performedusing anti-SURx antibodies. The inner zone of the gliotic capsuleimmediately outside of the gelatin sponge (gf in FIG. 22) was stronglylabeled with anti-SUR1 antibody (FIG. 22A) but not with anti-SUR2antibody (FIG. 22B). Although individual cells were not discerned at lowmagnification, higher magnification showed that SUR1 label was uniformlydistributed in individual cells after isolation (FIG. 22C).

Evidence for transcription of SUR1, but not SUR2 was also found inRT-PCR experiments run on mRNA from gelatin sponges isolated 7 daysafter implantation. The signal observed in astrocytes (FIG. 22D, lane 3)was present at the appropriate position on the gel, similar to that fromcontrol insulinoma RIN-m5f cells (FIG. 22D, lane 2). By contrast, mRNAfor SUR2 is not transcribed in reactive astrocytes (FIG. 22D, lane 5)although it is in cardiomyocytes used as control (FIG. 22D, lane 4).

Example 22 Characterization of the Inner Zone of the Gliotic Capsule

To examine whether or not all GFAP-positive reactive astrocytes in thegliotic capsule are SUR1 positive, brains from rats that had beenimplanted 1 week earlier with a gelatin sponge, then perfusion-fixed andequilibrated in 40% sucrose in PBS ×2 days were studied. Cryostatsections were double labeled with anti-GFAP and anti-SUR1 antibodies andstudied with immunofluorescence. For this and other immunolabelingexperiments, standard control protocol included use of the appropriateimmunogenic peptide when available or omission of primary antibody.

Five animals were sectioned and imaged with low power images. The imagesinvariably showed that the depth (thickness) of the GFAP response fromthe edge of the gelatin sponge was several-fold greater than the depthof the SUR1 response. Measurements of the depth of the GFAP responseyielded values of about 400-500 μm (FIG. 23A; in FIGS. 23A-23I, thelocation of the gelatin sponge implant was always to the left; bar inFIG. 23F equals 100 μm). By contrast, the prominent portion of the SUR1response extended for a depth of only 25-50 μm (FIG. 23D). Outside ofthe SUR1-positive zone was a wide region of GFAP-positive reactiveastrocytes that were mostly SUR1 negative. The SUR1 response was alwayslocated precisely at the interface with the foreign body, in theinnermost zone of the gliotic capsule. Cells that were SUR1 positivewere always GFAP positive. It was evident from this experiment thatcells clinging to the gelatin sponge and that were harvested with itwere likeliest to express SUR1. Also, it was clear that R1 astrocytes inthis innermost region comprised a unique subpopulation of reactiveastrocytes. From this observation emerged the concept of the “innerzone” of the gliotic capsule as being a unique entity, distinct from theremainder of the gliotic capsule.

Example 23 Other Characteristics of the Inner Zone of the GlioticCapsule

Other studies were performed to further evaluate the inner zone of thegliotic capsule. In previous experiments, it was found that primaryculture of R1 astrocytes under normoxic culture conditions resulted inloss of the SUR1-regulated NC_(Ca-ATP) channel after 3 days, whereascultured under hypoxic conditions resulted in continued expression ofthe channel (Chen et al., 2003). Thus, it was determined that expressionof the channel required hypoxic conditions, and thus the inner zone ofthe gliotic capsule where SUR1 expressing R1 astrocytes were found mightalso be hypoxic. To evaluate this, the histochemical marker,pimonidazole, was used which at pO₂<10 mm Hg, forms irreversiblecovalent adducts with cellular proteins that can be detectedimmunohistochemically (Arteel et al, 1998; Hale et al., 2002; Kennedy etal., 1997).

Briefly, rats were prepared with a stab injury and implantation of agelatin sponge. Rats were allowed to survive 1 week. Pimonidazole wasadministered prior to death, and cryosections were processed forimmunofluorescence study using the appropriate antibody to detectpimonidazole adducts. Cryosections were double labeled for GFAP. Thisexperiment confirmed the presence of hypoxic conditions restricted tothe SUR1-positive inner zone of the gliotic capsule, with the mostprominent pimonidazole labeling extending only 20-50 μm deep (FIG. 23B;GFAP not shown but the depth of the GFAP response resembled that in FIG.23A). High resolution imaging showed that pimonidazole labeling (FIG.23G, upper right) was present in large GFAP-positive astrocytes (FIG.23G, lower left).

It was reasoned that hypoxia of the inner zone might lead toup-regulation/activation of the hypoxia-responsive transcription factor,HIF-1. To examine this, immunolabeling was performed of sections withanti-HIF-1α antibodies with co-labeling for GFAP. This experimentconfirmed that HIF-1a labeling was mostly restricted to theSUR1-positive inner zone of the gliotic capsule, with labeling extendingonly 20-50 μm deep (FIG. 23C; GFAP not shown but the depth of the GFAPresponse resembled that in FIG. 23A). High resolution imaging showedthat HIF-1α labeling (FIG. 23H, upper right) was present in largeGFAP-positive astrocytes (FIG. 23H, lower left).

Expression of tight junction proteins was also examined Two tightjunction proteins, ZO-1 and occludin-5, were studied, labeling alternatecryosections with antibodies directed against these proteins. Sectionswere double labeled for GFAP. Again, only the innermost layer 20-50 μmdeep was labeled for either ZO-1 or occludin-5 (FIGS. 23E and 23F; GFAPnot shown but the depth of the GFAP response resembled that in FIG.23A). High resolution imaging showed that occludin-5 labeling (FIG. 23I,upper right) was present in large GFAP-positive astrocytes (FIG. 23I,lower left).

Thus, the inner zone of the gliotic capsule, with its R1 astrocytes thatexpress SUR1-regulated NC_(Ca-ATP) channels and tight junction proteins,may be acting as an important barrier between the foreign body and thebrain, e.g., a foreign body-brain barrier (FbBB). If true, one wouldexpect that breaching the barrier might significantly affect the overallresponse to injury.

Example 24 Manipulation of the Inner Zone

Rats were prepared with a stab injury and implantation of a gelatinsponge according to our usual protocol and were allowed to survive 1week. At time of surgery, rats were also implanted with osmoticmini-pumps subcutaneously with the delivery catheter placed in the brainat the site of injury. Animals received pumps with either glibenclamide(1 μM at 0.5 μl/hr×7 days) or diazoxide (10 μM at 0.5 μl/hr×7 days). Nosystemic toxicity was observed, neurological behavior was not impaired,and animals appeared healthy and were not febrile.

Cryosections of injured brains were examined for GFAP. In animalsreceiving glibenclamide, a well defined gliotic capsule was visualizedthat was sharply demarcated from surrounding brain, with the inner zoneappearing to be densely populated by GFAP-positive cells (FIG. 24A;gelatin sponge to the right). By contrast, animals receiving diazoxideshowed an expanded GFAP-positive response that extended farther from theforeign body, with an outer region that was poorly demarcated, and aninner zone that was loose and not compact (FIG. 24B; gelatin sponge tothe right).

Cryosections were also examined with the nuclear label, DAPI. Insections from glibenclamide-treated animals, most of the labeling wasattributable to GFAP-positive astrocytes. However, in sections fromdiazoxide-treated animals, DAPI labeling showed “sheets” of smallnucleated cells (dull spots in FIG. 25A). On inspection, these sheets ofcells appeared to be polymorphonuclear leukocytes (PMNs, neutrophils).This was confirmed by labeling with MMP-8, a PMN-specific marker (FIG.25B). It is important to note that no evidence of infection was present,and microbiological cultures of explanted materials showed no bacterialgrowth, including aerobic and anaerobic cultures, indicating that theinflammatory response was not due to infection.

Thus, protecting inner zone R1 astrocytes with glibenclamide appeared tohave restrained the overall GFAP-response to injury, whereas killinginner zone R1 astrocytes with diazoxide appeared to have caused anexpansion of the overall GFAP-response and recruitment of tremendousnumbers of neutrophils. These observations strongly reinforced theconcept of the “inner zone” of the gliotic capsule as being a uniqueentity, with a critical function in determining the overall response toinjury.

Example 25 SUR1 in Multiple Brain Pathologies

Tissues were obtained from the 3 rat models (trauma, abscess and stroke)and from the gliotic capsule surrounding human metastatic tumor, anddouble immunolabeling was performed with antibodies directed againstGFAP and SUR1. Low power views showed a layer of tissue adjacent to thegelatin sponge implant with positive immunolabeling for GFAP thatcoincided with positive immunolabeling for SUR1 (FIG. 26A,B).Examination of individual cells at high power showed that the SUR1immunolabel was present in large stellate-shaped astrocytes, confirmingthe presence of SUR1-positive R1 astrocytes in the inner zone of thegliotic capsule surrounding a foreign body implant (FIG. 26C).

A brain abscess model in the rat was studied. The abscess was producedby implanting an autologous fecal pellet subcortically under generalanesthesia. These animals survived quite well, although they showedevidence of mild weight loss. When sacrificed 1 week after surgery, apurulent cavity was found surrounded by a gliotic capsule. Low powerviews of the gliotic capsule adjacent to the area of puss showed cellswith positive immunolabeling for GFAP that coincided with positiveimmunolabeling for SUR1 (FIG. 26D,E). Examination of individual cells athigh power showed that the SUR1 immunolabel was present in largestellate-shaped astrocytes, confirming the presence of SUR1-positive R1astrocytes in the inner zone of the gliotic capsule surrounding brainabscess (FIG. 26F).

A standard stoke model in the rat was studied. The stroke was producedby intra-carotid insertion of a thread up to the bifurcation of theinternal carotid artery, placed under general anesthesia. Animalssurviving the stroke were sacrificed at 1 week and the brain wasexamined. Low power views of tissues adjacent to the area of strokeshowed cells with positive immunolabeling for GFAP that coincided withpositive immunolabeling for SUR1 (FIG. 26G,H). Examination of individualcells at high power showed that the SUR1 immunolabel was present inlarge stellate-shaped astrocytes, confirming the presence ofSUR1-positive R1 astrocytes in the gliotic capsule surrounding stroke(FIG. 26I).

Tissue was obtained from humans undergoing surgery for resection ofmetastatic brain tumors. At surgery, the gliotic capsule that surroundsthe metastasis is readily distinguished from the tumor itself and fromedematous white matter. Low power views of the gliotic capsule adjacentto the metastasis showed cells with positive immunolabeling for GFAPthat coincided with positive immunolabeling for SUR1 (FIG. 26J,K).Examination of individual cells at high power showed that the SUR1immunolabel was present in large stellate-shaped astrocytes withmultiple well-developed processes, confirming the presence ofSUR1-positive R1 astrocytes in the gliotic capsule surroundingmetastatic brain tumor in humans (FIG. 26L).

These data show for the first time SUR1 up-regulation in reactiveastrocytes at the site of formation of a gliotic capsule consistent withexpression of SUR1-regulated NC_(Ca-ATP) channels in R1 astrocytes. Thedata indicate that SUR1 expression in R1 astrocytes in the glioticcapsule was a common phenomenon in numerous pathological conditions thataffect the brain. These data highlight a unique opportunity tomanipulate R1 astrocytes of the inner zone selectively by exploitingpharmacological agents that act at SUR1 and that can therefore determinedeath or survival of these cells.

Overall, these observations strongly reinforced the concept of the“inner zone” of the gliotic capsule as being a unique entity, distinctfrom the remainder of the gliotic capsule.

Example 26 The NC_(ca-Atp) Channel and Necrotic Death

NC_(Ca-ATP) channels were studied in a rodent model of stroke. In thepenumbra, SUR1 labeling was found in stellate-shaped cells (FIG. 27A)that were also GFAP-positive. In the middle of the stroke, stellatecells were absent, but SUR1 labeling was found in round cells exhibitinga bleb-like appearance (FIG. 27B,C) that were also GFAP-positive (notshown). The round cells with blebbing in situ resembled reactiveastrocytes in vitro undergoing necrotic death after exposure to Naazide. The effect of glibenclamide vs. saline was determined.Glibenclamide or saline was administered via subcutaneously-implantedosmotic mini-pump (1 μM at 0.5 μl/hr). In saline treated rats, 3-daymortality after stroke was 68%, whereas in glibenclamide-treated rats,3-day mortality was reduced to 28% (n=29 in each group; p<0.001, by χ²).In separate animals, the stroke hemisphere in glibenclamide-treated ratscontained only half as much excess water as in saline-treated rats (n=5in each group; p<0.01, by t-test), confirming an important role of theNC_(Ca-ATP) channel in edema formation.

SUR1 was also studied in a rodent model of trauma. The effect of directinfusion of drugs into the site of trauma was examined using animplanted osmotic mini-pump. The channel inhibitor, glibenclamide, wasused to reduce death of reactive astrocytes, and the channel activator,diazoxide, to promote astrocyte death. Glibenclamide infusion reducedthe overall injury response, stabilized the gliotic capsule around theforeign body implant, and minimized the inflammatory response comparedto control.

Conversely, diazoxide essentially destroyed the gliotic capsule andincited a huge inflammatory response, characterized by massive influx ofpolymorphonuclear cells (PMNs) (FIG. 25A, B). These data suggested thatNC_(Ca-ATP) channel plays a critical role in the injury response, andthey strongly support the hypothesis that inflammation is closely linkedto activity of the NC_(Ca-ATP) channel and necrotic death of reactiveastrocytes.

Example 27 Permanent MCA Models

Adult male or female Wistar rats (275-350 gm) were fasted overnight thenanesthetized (Ketamine, 60 mg/kg plus Xylazine, 7.5 mg/kg, i.p.). Theright femoral artery was cannulated, and physiological parameters,including temperature, pH, pO₂, pCO₂ and glucose were monitored. Using aventral cervical incision, the right external carotid andpterygopalatine arteries were ligated. The common carotid artery wasligated proximally and catheterized to allow embolization of theinternal carotid artery.

For thromboembolic (TE) stroke, 7-8 allogeneic clots, 1.5 mm long, wereembolized. Allogeneic, thrombin-induced, fibrin-rich blood clots wereprepared (Toomy et al., 2002).

For large MCA strokes with malignant cerebral edema (MCE), the inventorsfirst embolized microparticles (Nakabayashi et al., 1997) [polyvinylalcohol (PVA) particles; Target Therapeutics, Fremont Calif.; 150-250 μmdiameter, 600 μg in 1.5 ml heparinized-saline], followed by standardpermanent intraluminal suture occlusion (Kawamura et al., 1991) using amonofilament suture (4-0 nylon, rounded at the tip and coated withpoly-L-lysine) advanced up to the ICA bifurcation and secured in placewith a ligature.

After stroke, animals are given 10 ml glucose-free normal saline bydermoclysis. Rectal temperature was maintained at ≈37° C. using aservo-controlled warming blanket until animals awoke from anesthesia.Blood gases and serum glucose at the time of stroke were: pO₂, 94±5 mmHg; pCO₂, 36±5 mm Hg; pH, 7.33±0.01; glucose 142±6 mg/dl in controls andpO₂, 93±3 mm Hg; pCO₂, 38±2 mm Hg; pH, 7.34±0.01; glucose 152±7 mg/dl inglibenclamide-treated animals.

With both models, animals awoke promptly from anesthesia and movedabout, generally exhibited abnormal neurological function, typicallycircling behavior and hemiparesis. Mortality with the thromboembolic(TE) model was minimal, whereas with the malignant cerebral edema (MCE)model, animals exhibited delayed deterioration, often leading to death.Most deaths occurred 12-24 hr after MCA occlusion, with necropsiesconfirming that death was due to bland infarcts. Rarely, an animal died<6 hr after stroke and was found at necropsy to have a subarachnoidhemorrhage, in which case it was excluded from the study. Mortality inuntreated animals with MCE and bland infarcts was 65%, similar to thatin humans with large MCA strokes (Ayata & Ropper, 2002).

Example 28 Studies on Stroke Size, Mortality, Tissue-Water, and DrugLocalization

After MCA occlusion (both TE and MCE models), mini-osmotic pumps (Alzet2002, Durect Corporation, Cupertino, Calif.) were implantedsubcutaneously that delivered either saline or glibenclamide (Sigma, St.Louis, Mo.; 300 μM or 148 μg/ml, 0.5 μl/hr subcutaneously, no loadingdose). Stroke size (TE model), measured as the volume of TTC(−) tissuein consecutive 2 mm thick slices and expressed as the percent ofhemisphere volume, was compared 48 after stroke in 2 treatment groups,each comprised of 10 male rats, treated with either saline orglibenclamide. Mortality (MCE model) was compared during the first weekafter stroke in 2 treatment groups, each comprised of 29 rats (19 femaleplus 10 male), treated with either saline or glibenclamide. Edema (MCEmodel) was compared at 8 hr after stroke in 2 treatment groups, eachcomprised of 11 male rats, treated with either saline or glibenclamide;rats in each of these 2 treatment groups were subdivided into 2subgroups, with the first of these being used to analyze water in theentire involved hemisphere (no TTC processing), and the second beingused to analyze water in the TTC(+) vs. TTC(−) portions of the involvedhemisphere. For localization of fluorescent-tagged drug, 20 male ratswere subjected to MCA stroke (MCE model) and were implanted withmini-osmotic pumps that delivered BODIPY-conjugated glibenclamide(BODIPY-FL-glyburide, Molecular Probes, Eugene, Oreg.; 300 μM or 235μg/ml, 0.5 μl/hr subcutaneously, no loading dose). Of these, 15 ratswere used for validation of drug action (mortality, tissue water andglucose) and 5 rats were used for determination of drug distribution.

Example 29 Immunolabeling

Brains were perfusion-fixed (4% paraformaldehyde) and cryoprotected (30%sucrose). Cryosections (10 μm) were prepared and immunolabeled usingstandard techniques (Chen et al., 2003). After permeabilizing (0.3%Triton X-100 for 10 min), sections were blocked (2% donkey serum for 1hr; Sigma D-9663), then incubated with primary antibody directed againstSUR1 (1:300; 1 hr at room temperature then 48 h at 4° C.; SC-5789; SantaCruz Biotechnology). After washing, sections were incubated withfluorescent secondary antibody (1:400; donkey anti-goat Alexa Fluor 555;Molecular Probes, OR). For co-labeling, primary antibodies directedagainst NeuN (1:100; MAB377; Chemicon, CA); GFAP (1:500; CY3 conjugated;C-9205; Sigma, St. Louis, Mo.) and vWf (1:200; F3520, Sigma) were usedand tissues were processed according to manufacturers' recommendations.Species-appropriate fluorescent secondary antibodies were used asneeded. Fluorescent signals were visualized using epifluorescencemicroscopy (Nikon Eclipse E1000).

Example 30 TTC Staining, Stroke Size

Freshly harvested brains were cut into 2-mm thick coronal sections, andslices were exposed to TTC (0.125% w/v in 62.5 mM Tris-HCl, 13 mM MgCl₂,1.5% dimethylformamide) for 30 min at 37° C. For stroke size, stainedsections were photographed and images were analyzed (Scion Image) todetermine the percent of the involved hemisphere occupied by TTC(−)tissue; no correction for edema was performed. For some determinationsof water or SUR1 protein content, individual coronal sections weredivided under magnification into 3 parts: (i) the non-involved, controlhemisphere; (ii) the TTC(+) portion of the involved hemisphere; (iii)the TTC(−) portion of the involved hemisphere. For each animal, pooledtissues from the 3 parts were then processed for tissue watermeasurements or for Western blots.

Example 31 Tissue Water Content

Tissue water was quantified by the wet/dry weight method (Hua et al.,2003). Tissue samples were blotted to remove small quantities ofadsorbed fluid. Samples were weighed with a precision scale to obtainthe wet weight (WW), dried to constant weight at 80° C. and low vacuum,and then reweighed to obtain the dry weight (WD). The percent H₂O ofeach tissue sample was then calculated as (WW−WD)×100/WW.

Example 32 Immunoblots

Tissues lysates and gels were prepared (Perillan et al., 2002).Membranes were developed for SUR1 (SC-5789; Santa Cruz Biotechnology),Kir6.1 (Santa Cruz) or Kir6.2 (Santa Cruz). Membranes were stripped andre-blotted for β-actin (1:5000; Sigma, St. Louis, Mo.), which was usedto normalize the primary data. Detection was carried out using the ECLsystem (Amersham Biosciences, Inc.) with routine imaging andquantification (Fuji LAS-3000).

Example 33 In Situ Hybridization

Non-radioactive digoxigenin-labeled probes were made according to themanufacturer's protocol (Roche) using SP6 or T7 RNA polymerase. RNAdig-labeled probes (sense and anti-sense) were generated from pGEM-Teasy plasmids (Promega) with the SUR1 insert (613 bp) flanked by theprimers: 5′ AAGCACGTCAACGCCCT 3′ (forward; SEQ ID NO: 1); 5′GAAGCTTTTCCGGCTTGTC 3′ (reverse; SEQ ID NO: 2). Fresh-frozen (10 μm) orparaffin-embedded (4 μm) sections of rat brain (3, 6, 8 hours after MCAstroke) were used for in situ hybridization (Anisimov et al., 2002).

Example 34 Inner Zone of the Gliotic Capsule

To assess if other causes of hypoxia, for example arterial occlusion,resulted in up-regulation of SUR1, two rodent models of permanent focalcerebral ischemia as described in the examples were used.

The MCE model was used to evaluate SUR1 protein and mRNA, and to assesseffects of SUR1 inhibition on edema and survival, while the TE model wasused to measure effects of SUR1 inhibition on stroke size. Absence ofperfusion (FIG. 29A), TTC staining (Mathews et al., 2000) (FIG. 29B) andGFAP immunolabeling were used to distinguish infarct from peri-infarctregions.

SUR1 expression increased transiently in the core of the infarct. Here,an increase in SUR1 became evident as early as 2-3 hr after MCAocclusion (FIG. 29D), well before onset of necrosis, and laterdisappeared as necrosis set in (FIG. 29C, right side of figure). Atthese early times before necrosis, SUR1 was very prominent in neuronsthat co-labeled with NeuN (FIG. 29D-F).

In pen-infarct regions, including the classical ischemic “watershed”zone between anterior cerebral artery (ACA) and MCA territories, SUR1expression increased later than in the core but was sustained. By 6-12hr, SUR1 expression sharply demarcated infarct and pen-infarct areas(FIG. 29C). Here, SUR1 expression was found in neurons, astrocytes andcapillary endothelial cells, as shown by co-labeling with NeuN, GFAP(FIG. 29G-I) and von Willebrand factor (FIG. 29J-L), respectively. SUR1is not normally expressed in such abundance in these cortical andsubcortical areas (Treherne & Ashford, 1991; Karschin et al., 1997) asis evident in contralateral tissues (FIG. 29C, left side of figure).

Western blots showed an increase in expression of SUR1 protein, mostprominently in pen-infarct regions (FIG. 30A-D). However, thepore-forming subunits of K_(ATP) channels, Kir6.1 or Kir6.2, were notup-regulated (FIG. 30C-D). In situ hybridization showed SUR1 transcriptsin neurons and capillaries from regions of ischemia that were notpresent in control tissues (FIG. 30E-G), suggesting that SUR1, but notK_(ATP) channels, was transcriptionally up-regulated in cerebralischemia.

Thus, these data suggest that SUR1, but not Kir6.1 or Kir6.2, istranscriptionally up-regulated in cerebral ischemia, first in regionsthat are destined to undergo necrosis, and later in peri-infarctregions.

Example 35 SUR1 Up-Regulation

FIG. 30A-G discussed in Example 34 showed that SUR1 was significantlyup-regulated in stroke. It also showed that the pore-forming subunits,Kir6.1 and Kir6.2, were not up-regulated in stroke, suggesting thatK_(ATP) channels were not involved. To prove that SUR1 up-regulation isdue to NC_(Ca-ATP) channels and not to K_(ATP) channels, patch clamprecordings of neurons and endothelial cells from ischemic regions wereperformed. Large neuron-like cells were enxymatically isolated 3-hr(FIG. 31A) and 6-hr after stroke. Patch clamp study was carried outusing Cs⁺ in the bath and pipette, to block all K⁺ channels includingK_(ATP) channels. These experiments showed robust cation channelactivity that was blocked by glibenclamide, as predicted for theNC_(Ca-ATP) channel (FIG. 31B). In addition, when channel activity wasrecorded with K⁺, the slope conductance was 34 pS (FIG. 31C,D), aspreviously reported in freshly isolated R1 astrocytes, and much lessthan the 70-75 pS reported for KATP channels.

Example 36 Function of SUR1 in Cerebral Ischemia

To determine the function of SUR1 that was newly expressed in cerebralischemia, the effects of glibenclamide, a highly selective inhibitor ofSUR1 was studied. The effect of glibenclamide on mortality (MCE model)was studied. In a large group of animals, both male and female,treatment with glibenclamide resulted in a dramatic reduction inmortality compared to saline, from 65% to 24% (p<0.002; FIG. 32A).

Since glibenclamide had been shown to ameliorate cytotoxic edema ofastrocytes induced by energy depletion (Chen et al., 2003), it wasreasoned that the beneficial effect on mortality was related to edema.The effect of glibenclamide on the formation of edema 8 hr afterinduction of stroke (MCE model) was examined. This is a time thatpreceded death of any animal in the mortality study. In the first of twoexperiments, water content in the involved and uninvolved hemisphereswas measured using the methods described above. For the controlhemisphere, water was 77.9±0.2%. For the involved hemisphere, water roseby 3.4%, to 81.3±0.5% for the group treated with saline, whereas it roseby only 2.0%, to 79.9±0.3%, for the group treated with glibenclamide.These values were significantly different (p<0.05), consistent with animportant role of SUR1 in formation of edema.

Next, to better characterize the location of edema, the water contentafter dividing coronal brain sections into viable TTC(+) and non-viableTTC(−) parts was examined. Water in the uninvolved hemisphere was78.0±0.1% (FIG. 32B), similar to the previous value of 77.9±0.2%,indicating that TTC processing had not altered water content. For theinvolved hemisphere, water in the TTC(+) tissue rose by 5.4%, to83.4±1.1% for the group treated with saline, whereas it rose by only2.5%, to 80.5±0.3%, for the group treated with glibenclamide (FIG. 32B).These values were significantly different (p<0.05). By contrast, valuesfor water in TTC(−) tissues, 78.7±1.0% and 78.6±0.4% with saline andwith glibenclamide, respectively, were not different (p=0.97), and wereonly slightly higher than the value for the uninvolved hemisphere(78.0%), reflecting a need for ongoing blood flow to increase tissuewater (FIG. 32B) (Ayata & Ropper, 2002).

In these animals, serum glucose at 8 hr when edema was measured remainedin a range unlikely to have an effect on ischemia-induced damage (Li etal., 1994; Wass & Lanier, 1996) (122±4 vs. 93±3 mg/dl for saline andglibenclamide-treated animals, respectively; 11 rats/group). Together,these data indicated that the edema was located almost entirely inviable peri-infarct (penumbral) tissue adjacent to the early core of thestroke, and that glibenclamide was highly effective in reducing it,consistent with an important role for SUR1 in formation of edema.

Thus, the data with low-dose glibenclamide, which is highly selectivefor SUR1 (Gribble & Reimann, 2003; Meyer et al., 1999) providedcompelling evidence of a critical role for SUR1 in formation of cerebraledema.

Example 37 The Effect of Stroke Size

A non-lethal thromboembolic (TE) model was used to assess stroke size 48hr after induction of stroke.

With the TE model, glibenclamide treatment resulted in a highlysignificant reduction in stroke volume, compared to saline controls(32.5±4.9% vs. 15.5±2.3%; p<0.01) (FIG. 32C-E). Essentially all animals,regardless of treatment group, suffered infarctions involving the basalganglia, which were supplied by terminal lenticulostriate arterioles.However, reduced stroke volumes in the glibenclamide group were oftenassociated with marked sparing of the cerebral cortex (FIG. 32C-D), aphenomenon previously reported with decompressive craniectomy (Doerfleret al., 2001). With glibenclamide, cortical sparing may reflect improvedleptomeningeal collateral blood flow due to reduced cerebral edema andreduced intracranial pressure.

Example 38 MCE Model Following Stroke

The fluorescent derivative, BODIPY-glibenclamide, was used to labeltissues in vivo following stroke (MCE model).

When delivered in the same manner as the parent compound, thefluorescent derivative exhibited similar protective effects, but wasless potent [7-day mortality, 40% (n=10); water in the TTC(+) portion ofthe involved hemisphere at 8 hr, 82.7±1.4% (n=5); serum glucose, 109±4mg/dl], consistent with reduced efficacy of the labeled drug (Zunkler etal., 2004). The low systemic dose of drug used yielded minimal labelingin the uninvolved hemisphere (FIG. 33B) and pancreas, and none in theunperfused core of the stroke. However, cells in pen-infarct regionswere clearly labeled, with well-defined labeling of large neuron-likecells and of microvessels (FIG. 33A), including capillaries (FIG. 33C),that showed prominent expression of SUR1 (FIG. 33D). Preferentialcellular labeling in ischemic brain likely reflected not only anincrease in glibenclamide binding sites, but also an increase in uptake,possibly due to alteration of the blood brain barrier.

Thus, the data indicated the presence of NC_(Ca-ATP) channels incapillary endothelium and neurons in addition to their previouslydescribed presence in astrocytes (Chen et al., 2001; Chen et al., 2003).Additional patch clamp experiments on neurons and microvessels isolatedfrom ischemic cortex 1-6 hr after MCA occlusion (MCE model) confirmedthe presence of NC_(Ca-ATP) channels, showing a non-selective cationchannel of around 30-35 pS conductance, that was easily recorded withCs⁺ as the charge carrier, and that was blocked by glibenclamide. Thischannel was not present in cells from non-ischemic cerebral tissues.

In view of the above, it is suggested that SUR1-regulated NC_(Ca-ATP)channels that are opened by ATP depletion and that are newly expressedin ischemic neurons, astrocytes and endothelial cells constitute animportant, heretofore unidentified pathway for Na⁺ flux required forformation of cytotoxic and ionic edema. Together, these findings suggesta critical involvement of SUR1 in a new pathway that determinesformation of edema following cerebral ischemia. Molecular therapiesdirected at SUR1 may provide important new avenues for treatment of manytypes of CNS injuries associated with ischemia.

Example 39 Co-Administration of Glibenclamide and tPA

A rodent model of thromboembolic stroke was used (Aoki et al., 2002;Kijkhuizen et al., 2001; Kano et al., 2000; Sumii et al., 2002; Tejimaet al., 2001). Briefly, male spontaneously hypertensive rats that havebeen fasted overnight are anesthetized using halothane (1-1.5% in a70/30 mixture of N₂O/O₂) with spontaneous respiration (Lee et al., 2004;Sumii et al., 2002). Rectal temperature was maintained at ≈37° C. with athermostat-controlled heating pad. The right femoral artery wascannulated, and physiological parameters, including temperature, meanblood pressure, pH, pO₂, and pCO₂, glucose were monitored. Temporaryfocal ischemia was obtained with an embolic model that used allogeneicclots to occlude the MCA. Allogeneic, thrombin-induced, fibrin-richblood clots were prepared using methods adapted from Niessen et al.(Asahi et al., 2000; Niessen et al., 2003; Sumii et al., 2002). Sevenclots, 1.5 mm long, were used for embolizing.

Using a ventral cervical incision, the internal and external carotidarteries were exposed. The external carotid artery and pterygopalatinearteries were ligated. Removable surgical clips were applied to thecommon and internal carotid arteries. The modified PE-50 cathetercontaining the clots was inserted retrograde into the external carotidartery and advanced up to the internal carotid artery. The temporaryclips were removed, and the clots were injected. Incisions were closed.

After stroke, animals were given glucose-free normal saline, 10 mltotal, by dermoclysis. Temperature was maintained until animals wereawake and were moving about.

Just prior to the time designated for treatment (reperfusion), animalswere re-anesthetized and the femoral vein was cannulated. At the timedesignated for treatment, saline, or a loading dose of glibenclamide(1.5 μg/kg, i.v., Sigma, St. Louis) was first administered. Then,reperfusion was achieved with i.v. administration of rtPA (10 mg/kg,Alteplase, Genetech; dissolved in 2 ml distilled water, given over 30min) (Buesseb et al., 2002). Then, using a dorsal thoracic incision, amini-osmotic pump (Alzet 2002, Durect Corporation, Cupertino, Calif.)was implanted subcutaneously that delivered either saline orglibenclamide (300 μM or 148 μg/ml, 0.5 μl/hr s.q.). Physiologicalparameters, including temperature, mean blood pressure (tail cuffplethysmography), blood gases and glucose were monitored.

At the same time of 6 hr, animals were co-treated with either saline orglibenclamide (loading dose of 1.5 μg/kg i.v. plus implantation of amini-osmotic pump containing 148 μg/ml=300 μM delivered at ½ μl/hr).Animals were euthanized 24 hr following stroke and brains were perfusedto remove blood from the intravascular compartment. Coronal sections ofthe fresh brains were prepared and photographed, following whichsections were processed for TTC staining to identify areas ofinfarction.

All animals (5/5) co-treated with saline showed large regions ofhemorrhagic conversion in cortical and subcortical parenchymal areas ofinfarction, along with evidence of intraventricular hemorrhage (FIG.34A-D). In contrast, only 1/5 animals co-treated with glibenclamide hadhemorrhagic conversion, with 4/5 showing no evidence of hemorrhage (FIG.34E-H).

These data suggest that there was protection from hemorrhagic conversionwith the administration of glibenclamide, as well as reduction in strokesize, ionic edema, and vasogenic edema.

Example 40 Isolation of Brain Capillaries and Endothelial Cells

The method was adapted in part from Harder et al. (1994) withmodifications as previously reported (Seidel, 1991). Briefly, a rat wasdeeply anesthetized, the descending aorta was ligated, the right atriumwas opened and the left ventricle was cannulated to allow perfusion of50 ml of a physiological solution containing a 1% suspension of ironoxide particles (particle size, 10 μm; Aldrich Chemical Co.). The brainwas removed, the pia and pial vessels were stripped away and thecortical mantel is minced into pieces 1-2 mm³ with razor blades. Thetissue pieces were incubated with trypsin plus DNAse and then sievedthrough nylon mesh (210 μm). Retained microvessels were resuspended incollagenase, agitated and incubated at 37° C. for an additional 10 min.To terminate the digestion, microvessels were adhered to the side of thecontainer with a magnet and washed repeatedly to remove enzyme andcellular debris.

Using these methods yielded healthy-appearing microvascular structuresthat were suitable for further digestion to obtain single cells (FIG.36) for further experiments.

Isolated endothelial cells were studied using freshly isolatedendothelial cells using a nystatin-perforated patch technique. Withphysiological solutions, the cells exhibited a prominent, stronglyrectifying inward current at negative potentials, and a modest outwardcurrent at positive potentials (FIG. 37A), yielding a characteristiccurrent-voltage curve with near-zero current at intermediate potentials(FIG. 37C), similar to previous observations in freshly isolatedendothelial cells (Hogg et al., 2002). When K⁺ in the pipette solutionwas replaced with Cs⁺, K⁺ channel currents were completely blocked. Inendothelial cells, this yielded a current-voltage curve that was linear(FIG. 37E). These data demonstrated that voltage dependent channels infreshly isolated endothelial cells are exclusively K⁺ channels that donot carry Na⁺.

Example 41 Isolation of Neurons

Neurons were isolated from vibratome sections Immunolabeling experimentsindicated that ischemic NeuN-positive neurons expressed SUR1 within 2-3hr after MCAO, before necrosis was evident. Therefore, tissues wereprepared at 2-3 hr after MCAO. The brain was divided coronally at thelevel of the bregma, and cryosections were prepared from one half andvibratome sections were prepared from the other half. Cryosections (10μm) were used for TTC staining (Mathews et al., 2000) or alternatively,high-contrast silver infarct staining (SIS), (Vogel et al., 1999) toidentify the region of ischemia, and for immunolabeling, to verify SUR1up-regulation in neurons double labeled for NeuN. Vibratome sections(300 μm) were processed (Hainsworth et al., 2000; Kay et al., 1986;Moyer et al., 1998) to obtain single neurons for patch clamping.Selected portions of coronal slices were incubated at 35° C. in HBSSbubbled with air. After at least 30 min, the pieces were transferred toHBSS containing 1.5 mg/ml protease XIV (Sigma). After 30-40 min ofprotease treatment, the pieces were rinsed in enzyme-free HBSS andmechanically triturated. For controls, cells from mirror-image corticalareas in the uninvolved hemisphere were used. Cells were allowed tosettle in HBSS for 10-12 min in a plastic Petri dish mounted on thestage of an inverted microscope. Large and medium-sized pyramidal-shapedneurons were selected for recordings. At this early time of 2-3 hr, onlyneurons and capillaries, not astrocytes, show up-regulation of SUR1.

Once the cells were isolated patch clamp experiments using well knownmethods including whole-cell, inside-out, outside-out and perforatedpatch were used (Chen et al., 2003; Chen et al., 2001; Perillan et al.,2002; Perillan et al., 2000; Perillan et al., 1999)

Example 42 MMP Inhibition by Glibenclamide

Activation of MMP-9 & MMP-2 in stroke tissue was compared to controls.Briefly, gelatinase activity of recombinant enzyme and stroke tissueunder control conditions (CTR), in presence of glibenclamide (10 μM),and in presence of MMP-inhibitor II (300 nM; Calbiochem).

Next, the supernatants underwent a gelatinase purification process withgelatin-Sepharose 4B (Pharmacia), and Zymography was performed on thepurified supernatants in sodium dodecyl sulfate gels containing gelatin(Rosenberg, 1994). Dried gels were scanned with a transparency scanner,and images were analyzed by densitometry. The relative lysis of anindividual sample was expressed as the integrated density value of itsband and divided by the protein content of the sample.

Zymography confirmed that gelatinase activity was increased after stroke(FIG. 35A), and showed that gelatinase activity assayed in the presenceof glibenclamide (FIG. 35B, Glibenclamide) was the same as that assayedwithout (FIG. 35B, CTR), although gelatinase activity was stronglyinhibited by commercially available MMP inhibitor II (FIG. 35B,MMP-2/MMP-9 inhibitor). These data demonstrated that glibenclamide didnot directly inhibit gelatinase activity, and suggested that thereduction of hemorrhagic conversion observed with glibenclamide likelycame about due to a beneficial, protective effect of glibenclamide onischemic endothelial cells.

Example 43 Up-Regulation of SUR1-mRNA in Stroke

Additional molecular evidence for involvement of SUR1 in stroke wasobtained using quantitative RT-PCR.

Total RNA was extracted and purified from samples of homogenized braintissues contralateral (CTR) and ipsilateral to MCAO (STROKE) usingguanidine isothyocyonatye. cDNA was synthesized with 4 μg of total RNAper 50 μl of reaction mixture using TaqMan RT kit (Applied Biosystems).Relative values of SUR1-mRNA were obtained by normalizing to Him(histone 1 member 0). The following probes were used SUR1 forward:GAGTCGGACTTCTCGCCCT (SEQ ID NO: 3); SUR1 reverse: CCTTGACAGTGGCCGAACC(SEQ ID NO: 4); SUR1 TaqMan Probe: 6-FAM-TTCCACATCCTGGTCACACCGCTGTTAMRA(SEQ ID NO: 5); H1f0 forward: CGGACCACCCCAAGTATTCA (SEQ ID NO: 6); H1f0reverse: GCCGGCACGGTTCTTCT (SEQ ID NO: 7); H1f0 TaqMan Probe:6-FAM-CATGATCGTGGCTGCTA TCCAGGCA-TAMRA (SEQ ID NO: 8).

These data showed that mRNA for SUR1 was significantly increased in thecore region, 3 hr after MCAO (FIG. 38).

Example 44 SUR1 Knockdown (SUR1KD) is Protective

To further test involvement of SUR1, SUR1 expression was “knocked down”in situ by infusing oligodeoxynucleotide (ODN) for 14 days using amini-osmotic pump, with the delivery catheter placed in the gelfoamimplantation site in the brain, in the otherwise standard model that theinventors use for R1 astrocyte isolation (Perillan et al., 1980,Perillan et al., 2002, Perillan et al., 2000, Perillan et al., 1999).Knockdown of SUR1 expression (SUR1KD) was achieved using antisense (AS;5′-GGCCGAGTGGTTCTCGGT-3′ (SEQ ID NO: 9)) (Yokoshiki et al., 1999)oligodeoxynucleotide (ODN), with scrambled (SCR;5′-TGCCTGAGGCGTGGCTGT-3′ (SEQ ID NO: 10)) ODN being used as control.

Immunoblots of gliotic capsule showed significant reduction in SUR1expression in SUR1 knockdown (SUR1KD) tissues compared to controlsreceiving scrambled sequence ODN (FIGS. 39A and 39B).

The inventors enzymatically isolated single cells from SUR1KD andcontrols using a standard cell isolation protocols described above (Chenet al., 2003) to assess functional responses to ATP depletion induced byNa azide. In R1 astrocytes from control tissues, Na azide (1 mM) causedrapid depolarization due to Na⁺ influx attributable to activation ofNC_(Ca-ATP) channels (FIG. 39C). Notably, this depolarizing response wasopposite the hyperpolarizing response observed when K_(ATP) channelswere activated. In R1 astrocytes from SUR1KD, however, Na azide hadlittle effect on resting membrane potential (FIG. 39D). In controls,application of Na azide resulted in depolarization of 64±3.7 mV, whereasin cells for SUR1KD, depolarization was only 8.7±1.7 mV (FIG. 39E).

In addition, membrane blebbing that typically follows exposure to Naazide was not observed in cells from SUR1KD, confirming the role forSUR1 in cytotoxic edema of R1 astrocytes.

Example 45 Molecular Factors that Regulate SUR1 Expression

Based on work in pancreatic β cells, a number of SP1 transcriptionfactor binding sites have been identified in the proximal SUR1 promoterregion that are considered to be important for activation of SUR1transcriptional activity (Ashfield et al., 1998; Hilali et al., 2004).Notably, SP1 has essentially not been studied in stroke (Salminen etal., 1995).

Briefly, the ischemic peri-infarct tissues was immunolabeled for SP1,which is important for SUR1 expression, for HIF1α, which is widelyrecognized to be up-regulated in cerebral ischemia (Semenza 2001; Sharpet al., 2000) and for SUR1 itself. SP1 was prominently expressed inlarge neuron-like cells and in capillaries (FIG. 40A, 40C) in regionsconfirmed to be ischemic by virtue of expression of HIF1α (FIG. 40B).Notably, capillaries that expressed SP1 also showed prominent expressionof SUR1 (FIG. 40C, 40D). Contralateral control tissues showed littleimmunolabeling for SP1 and none for HIF1α (FIG. 40E, 40F).

Nuclear SP1 localization was significantly augmented early-on in stroke(FIG. 41A, 41B), and nuclear SP1 was found in large neuron-like cellsthat express SUR1 following MCAO (FIG. 41C).

HIF1α knock-down animals were obtained by infusion of antisenseoligodeoxynucleotide at the site of gelfoam implant. FIG. 42 confirmsthe HIF1α knock-down animals results in a significant decrease in SUR1expression (FIG. 42B, 42D), providing strong evidence that not only SP1but also HIF1α is likely to be an important regulator of SUR1expression.

Example 46 Blood Flow in Peri-Infarct Tissue is Protected by Treatmentwith Nc_(ca-Atp) Channel Antagonist

Block of SUR1 by systemic administration of low-dose glibenclamidereduces cerebral edema, infarct volume and mortality, with the reductionin infarct volume being associated with cortical sparing. Athromboembolic (TE) stroke model associated with non-lethal infarctionsfrom MCAO was used to study effects on infarct volume in rats. Given thestriking effects on mortality and edema, the inventors sought todetermine whether glibenclamide would have a favorable effect on infarctvolume. This was not feasible with the MCE model because of the highincidence of early mortality. We therefore utilized a non-lethalthromboembolic (TE) model that would allow assessment of infarct volumeat 2 and 7 days after MCAO. At 2 days, glibenclamide treatment resultedin a highly significant reduction in infarct volume, compared to salinecontrols (35.5±4.4% vs. 16.7±2.2%; p<0.01). A similar observation wasmade at 7 days (15.2±1.2%; p<0.01), indicating again that the effect oftreatment was durable.

All animals, regardless of treatment group, suffered infarctionsinvolving the basal ganglia, which are supplied by terminal arterioles.However, reduced infarct volumes in the glibenclamide groups were oftenassociated with marked sparing of the cerebral cortex, a phenomenonpreviously reported with decompressive craniectomy. (Doerfler, et al.,(2001)). We hypothesized that cortical sparing with glibenclamide mightreflect improved leptomeningeal collateral blood flow, which could bedue to reduced cerebral edema. The effective dose of glibenclamide was75 ng/hr. Direct vasodilation was not expected, since glibenclamide isnormally vasoconstrictive due to block of KATP channels. (Lindauer, etal., (2003), and Tomiyama, et al., (1999)).

Blood flow was measured using laser Doppler flowmetry in order todetermine the effects of glibenclamide treatment on cerebral blood flow.Using the same TE model, measurements of relative cerebral blood flowwere obtained for somatosensory cortex supplied by the middle cerebralartery (MCA). Laser Doppler flowmetry showed values in the involvedhemisphere that were significantly reduced 1 hr after middle cerebralartery occlusion (MCAO) in both saline- and glibenclamide-treated groups(FIG. 43). However, flow measurements recovered completely by 48 hr inglibenclamide-treated animals but not in saline-treated animals (FIG.43), consistent with the cortical sparing observed.

Methods

Relative cerebral blood flow (TE model) was measured using laser Dopplerflowmetry (LDF) in 2 groups, each consisting of 4 male rats, treatedwith either saline or glibenclamide. Prior to MCAO, two 1.5-mm pits werecarefully drilled halfway through the skull over the left and rightsomatosensory cortex (MCA territory), 3 mm posterior and 3 mm lateral tothe bregma. A two-channel LDF instrument (DRT4; Moor Instruments,Axminster, UK) was used to simultaneously measure blood flow in bothhemispheres. LDF readings were normalized by adjusting the depth of thepits to obtain a ratio of blood flow of ˜1.0 between sides. Once thisratio had been obtained, five sets of LDF measurements were taken at 1min intervals, values for each location were averaged and the ratio ofipsilateral to contralateral LDF values was calculated. This techniqueminimized effects of intra-measurement differences in probe position,angle, lighting condition, etc. Once baseline CBF had been determined,skin incisions over the pits were closed and the procedure for MCAO wasinitiated. Relative CBF measurements were later repeated at 1 hr and 48hours after MCAO, using the same pits and the same method of averaging 5bilateral measurements obtained at 1-min intervals.

Edema (MCE model) was analyzed 8 hr after MCAO in 2 series of animals.In the first series, tissue water was analyzed in the uninvolved vs.involved hemisphere of 2 groups of 11 male rats, treated with eithersaline or glibenclamide (no TTC processing).

In the second series, tissue water was analyzed in the uninvolvedhemisphere and in the TTC(+) vs. TTC(−) portions of the involvedhemisphere in 3 groups of 6 male rats treated with either saline alone,vehicle (saline plus DMSO) or glibenclamide. Tissue water was quantifiedby the wet/dry weight method. Tissue samples were blotted to removesmall quantities of adsorbed fluid. Samples were weighed with aprecision scale to obtain the wet weight (W_(W)), dried to constantweight at 80° C. and low vacuum, and then reweighed to obtain the dryweight (W_(D)). The percent H₂O of each tissue sample was thencalculated as (W_(W)−W_(D))×100/W_(W).

Infarct volume (TE model), measured as the volume of TTC(−) tissue inconsecutive 2 mm thick slices and expressed as the percent of hemispherevolume, was compared in 3 treatment groups, consisting of 9, 9 and 7male rats, treated with saline and assessed at 2 days, or treated withglibenclamide and assessed at 2 days or 7 days after MCAO.

Permanent MCA occlusion (MCAO) models. This study was performed inaccordance with the guidelines of the Institutional Animal Care and UseCommittee. Adult male or female Wistar rats (275-350 gm) were fastedovernight then anesthetized (Ketamine, 60 mg/kg plus Xylazine, 7.5mg/kg, i.p.). The right femoral artery was cannulated, and physiologicalparameters, including temperature, pH, pO₂, pCO₂ and glucose weremonitored. Using a ventral cervical incision, the right external carotidand pterygopalatine arteries were ligated. The common carotid artery wasligated proximally and catheterized to allow embolization of theinternal carotid artery. For the thromboembolic (TE) stroke model, 7-8allogeneic clots, 1.5 mm long, were embolized. Allogeneic,thrombin-induced, fibrin-rich blood clots were prepared as described.For large MCA infarcts with malignant cerebral edema (MCE), theinventors first embolized microparticles [polyvinyl alcohol (PVA)particles; Target Therapeutics, Fremont Calif.; 150-250 μm diameter, 600μg in 1.5 ml heparinized-saline], followed by standard permanentintraluminal suture occlusion using a monofilament suture (4-0 nylon,rounded at the tip and coated with poly-L-lysine) advanced up to the ICAbifurcation and secured in place with a ligature. After MCAO, animalswere given 10 ml of glucose-free normal saline by dermoclysis. Rectaltemperature was maintained at about 37° C. using a servo-controlledwarming blanket until animals awoke from anesthesia. Blood gases andserum glucose at the time of MCAO were: pO₂, 94±5 mm Hg; pCO₂, 36±5 mmHg; pH, 7.33±0.01; glucose 142±6 mg/dl in controls and pO₂, 93±3 mm Hg;pCO₂, 38±2 mm Hg; pH, 7.34±0.01; glucose 152±7 mg/dl inglibenclamide-treated animals. With both models, animals awoke promptlyfrom anesthesia and moved about, generally exhibited abnormalneurological function, typically circling behavior and hemiparesis.Mortality with the TE model was minimal, whereas with the MCE model,animals exhibited delayed deterioration, often leading to death. Mostdeaths occurred 12-24 hr after MCAO, with necropsies confirming thatdeath was due to bland infarcts. Rarely, an animal died <6 hr after MCAOand was found at necropsy to have a subarachnoid hemorrhage, in whichcase it was excluded from the study. Mortality in untreated animals withMCE and bland infarcts was 65%, similar to that in humans with large MCAstrokes.

Within 2-3 min after MCAO (both TE and MCE models), mini-osmotic pumps(Alzet 2002, 14 day pump, 0.5 μl/hr; Durect Corporation, Cupertino,Calif.) were implanted subcutaneously that delivered either saline (0.9%NaCl), vehicle (saline plus DMSO) or glibenclamide in vehicle,subcutaneously (no loading dose). Glibenclamide (Sigma, St. Louis, Mo.)was prepared as a 10 mM stock solution in DMSO, with 15 μl stocksolution diluted into 500 μl saline to give a final concentration of 148μg/ml or 300 μM in the pump. The effective dose of glibenclamide was 75ng/hr. The effective dose of DMSO was 15 nl/hr, which is what wasdelivered in vehicle-treated animals.

TTC (triphenyltetrazolium chloride) staining was measured to determineinfarct volume. Freshly harvested brains were cut into 2-mm thickcoronal sections, and slices were exposed to TTC (0.125% w/v in 62.5 mMTris-HCl, 13 mM MgCl₂, 1.5% dimethylformamide) for 30 min at 37° C. Forinfarct volume, stained sections were photographed and images wereanalyzed (Scion Image) to determine the percent of the involvedhemisphere occupied by TTC(−) tissue; no correction for edema wasperformed. For some determinations of water content or SUR1 proteincontent, individual coronal sections were divided under magnificationinto 3 parts: (i) the uninvolved, control hemisphere; (ii) the TTC(+)portion of the involved hemisphere; (iii) the TTC(−) portion of theinvolved hemisphere. For each animal, tissues from the 3 parts were thenprocessed for tissue water measurements, or Western blots.

These findings indicate that the SUR1-regulated NC_(Ca-ATP) channel iscritically involved in development of cerebral edema, that modulation ofthe SUR1-regulated NC_(Ca-ATP) channel can lead to improved blood flowin peri-infarct tissue, and that targeting SUR1 provides an importantnew therapeutic approach to stroke.

Example 47 NC_(ca-Atp) Channel Antagonist Treatment Reduces Edema Evenwith Added Glucose Treatment

Although the dose of glibenclamide was low, a drop in serum glucoseconcentration in glibenclamide-treated animals was noted in theexperiments described above. The drop in glucose by glibenclamide raisedthe question whether the beneficial effect of glibenclamide on edema wasmediated directly via NC_(Ca-ATP) channels, or indirectly via reductionin serum glucose.

Tissue water as a measure of edema was measured in rats in a middlecerebral artery occlusion (MCAO) model of stroke. As in Example 46, theeffective dose of glibenclamide was 75 ng/hr delivered by subcutaneouslyimplanted Alzet mini-osmotic pump. Animals treated with glibenclamide(GLIB) alone experienced reduced serum glucose. For example, serumglucose concentration at 8 hr, when edema was measured, was 122±4 forsaline-treated animals (SALINE) vs. 93±3 mg/dl for glibenclamide-treatedanimals (GLIB) (see FIG. 44). Administration of glucose 4 hours afterocclusion resulted in serum glucose concentrations of 141±4 mg/dl at 8hours after occlusion.

Edema measurements in the same brain areas and at the same time inanimals treated with GLIB indicated that GLIB reduced edema irrespectiveof the glucose concentration. In these animals, supplemental glucose (1gm/kg, i.p.) was administered 4 hr after MCAO. This dose of glucose isreported to produce levels of hyperglycemia of 300 mg/dl, when measuredshortly after administration. Animals were sacrificed 8 hr after MCAOfor measurements of edema (FIG. 44, GLIB+GLUCOSE). Serum glucose 4 hrafter glucose administration (i.e., at time of sacrifice, 8 hr afterMCAO) was still elevated (141±4 mg/dl). However, in these animals, GLIBwas just as effective in reducing edema, even in the face ofhyperglycemia.

These results indicate that adding glucose does not impair theprotective effect of SUR1 antagonist treatment, and may enhance theprotective effect of SUR1 antagonist treatment.

Example 48 Delayed Treatment with Glibenclamide Reduces Stroke Volume inRats Following Middle Cerebral Artery Occlusion (MCAO)

Stroke volume in rats was measured as discussed above. Glibenclamide(3.3 μg/kg or 33.0 μg/kg) was given as indicated in FIG. 45. Animalstreated with the higher dose of glibenclamide were also given 1 gm/kgglucose in order to counteract hypoglycemia caused by the glibenclamide.

Stroke model: thromboembolic embolization of allogeneic clots viainternal carotid artery in male Wistar rats, 275-325 gm. Treatment:within 2-3 min after MCAO, animals were implanted with mini-osmoticpumps fitted with catheters of a length calibrated to delay onset ofdrug delivery by the amount of time indicated; the pumps were filledwith glibenclamide, 300 μM, that was delivered at a rate of 0.5 μl/hr,giving an effective infusion rate of 75 ng/hr for glibenclamide, and aneffective delivery rate of 15 nl/hr for DMSO (used as vehicle solvent);at the designated time, animals were also injected intraperitoneallywith a loading dose of glibenclamide, either 3.3 or 33 μg/kg, and in thecase of the higher dose of glibenclamide, with a supplemental dose ofglucose of 1 gm/kg. Stroke volume was determined at 48 after MCAO fromthe volume of TTC(−) tissue and is expressed as the percent ofhemisphere volume in FIG. 45. Values of “n” indicate the number of ratsper group; asterisks (*) indicates a statistically significant (P<0.05)difference in volume compared to saline (SAL) control as illustrated inFIG. 45.

A significant reduction in stroke volume was observed when glibenclamideinfusion was begun: (i) immediately after stroke, with no loading dose;(ii) 2 hr after stroke, with a loading dose of 3.3 μg/kg; (iii) and upto 6 hr after stroke with a loading dose of 33 μg/kg. Thus the lowerdose of glibenclamide (3.3 μg/kg) was effective at reducing strokevolume in experimental animals subjected to middle cerebral arteryocclusion (MCAO) when the glibenclamide was given at 0 or 2 hours afterMCAO. Although some reduction in stroke volume was seen at 4 hours afterMCAO with the lower dose of glibenclamide, the difference was notstatistically significant with this number of animals. However,statistically significant reductions in stroke volume (as compared tocontrol) were observed in animals treated with the higher dose ofglibenclamide (33.0 μg/kg, with co-administered glucose) given at 4 andat 6 hours after MCAO, as shown in FIG. 45. Thus, a larger dose of 33.0μg/kg was effective up to three times as long after MCAO as was thesmaller dose of glibenclamide.

These data indicate that the beneficial effect of glibenclamide can beobtained even with substantial delay in treatment, consistent with thebeneficial effect being due to a reduction in edema that permitsleptomeningeal collateral flow that helps salvage cortical structures.These data also demonstrate that co-administration of glibenclamide withglucose is effective in reducing stroke volume, that suchco-administration with glucose allows treatment with higher doses ofglibenclamide without the possibly deleterious effects of lowered bloodglucose, and allows for effective sulfonylurea treatment with greaterdelay before initiating treatment after stroke than appeared possiblewith lower sulfonylurea doses.

Example 49 Glibenclamide Reduces Hemorrhagic Conversion

Hemorrhagic conversion is a serious condition that often follows strokeor ischemic insult, in which reperfusion to ischemic tissue causesfurther damage to compromised tissue as anoxic and acidic fluids whichhad accumulated in non-perfused tissues flows to other tissues as bloodflow is restored to the region. Further, damage can come from leakyendothelial cells and blood vessels distal to the ischemic damage.Accordingly, an outcome study was designed as indicated to determine theeffect of glibenclamide on hemorrhagic conversion.

In this study, male rats of the spontaneously hypertensive (SHR) strainwere subjected to a thromboembolic stroke and then treated withtissueplasminogen activator (tPA) to dissolve the clot and restoreperfusion to non-perfused brain tissue. In particular, thromboembolicstroke was performed six hours after initiation of the experimentalstroke, tPA was administered intravenously (10 mg/kg over 30 min), alongwith either saline (control) or glibenclamide. Glibenclamide-treatedanimals were given a loading dose of 1.5 μg/kg intravenously (i.v.) anda sub-cutaneous (s c) mini osmotic pump was implanted that delivered 148μg/ml (equivalent to 300 μM at ½ μl/hr) to the animals.

The internal carotid artery (ICA) of male SHR rats were embolized withallogeneic thrombi to produced MCAO. Six hours later, animals weretreated with tPA (10 mg/kg i.v over 30 min) and co-treated with eithersaline or glibenclamide (1.5 mg/kg i.v. bolus plus implantation of as.c. pump that delivered a 300 mM solution at 0.5 ml/hr). At 24 hr afterstroke, brains were perfused to remove intravascular blood, sectionedcoronally, photographed, and processed for TTC staining. Results areshown in FIG. 46. Rows 1-2 (A-D) are from animals co-treated withsaline; rows 3-4 (E-H) are from animals co-treated with glibenclamide.The left column of photographs of coronal sections shows, in rows 1-2only, intraventricular hemorrhage, plus large areas of hemorrhagicconversion in ischemic cortical/subcortical regions (red areas on theright side of pictures; arrows). The right column of photographs ofTTC-processed sections from the same animals show the areas ofinfarction.

As shown in FIG. 46, the incidence of hemorrhage within the strokeregion (measured at 24 hours) was reduced by glibenclamide treatment ascompared with control. Although 5 of 6 animals co-treated with salineshowed hemorrhagic conversion, only 1 of 6 animals treated withglibenclamide showed hemorrhagic conversion, demonstrating the efficacyof glibenclamide treatment to reduce or prevent hemorrhagic conversionfollowing thromboembolic stroke. FIG. 46 thus demonstrates thatglibenclamide treatment reduces hemorrhagic conversion in tPA-treatedanimals, and extends the time window after ischemic insult within whichtPA may be administered without deleterious effects.

The foregoing disclosure of the preferred embodiments of the presentinvention has been presented for purposes of illustration anddescription. It is not intended to be exhaustive or to limit theinvention to the precise forms disclosed. Many variations andmodifications of the embodiments described herein will be apparent toone of ordinary skill in the art in light of the above disclosure. Thescope of the invention is to be defined only by the claims appendedhereto, and by their equivalents.

Further, in describing representative embodiments of the presentinvention, the specification may have presented the method and/orprocess of the present invention as a particular sequence of steps.However, to the extent that the method or process does not rely on theparticular order of steps set forth herein, the method or process shouldnot be limited to the particular sequence of steps described. As one ofordinary skill in the art would appreciate, other sequences of steps maybe possible. Therefore, the particular order of the steps set forth inthe specification should not be construed as limitations on the claims.In addition, the claims directed to the method and/or process of thepresent invention should not be limited to the performance of theirsteps in the order written, and one skilled in the art can readilyappreciate that the sequences may be varied and still remain within thespirit and scope of the present invention. Further, in describingrepresentative embodiments of the present invention, the specificationmay have presented the method and/or process of the present invention asa particular sequence of steps. However, to the extent that the methodor process does not rely on the particular order of steps set forthherein, the method or process should not be limited to the particularsequence of steps described. As one of ordinary skill in the art wouldappreciate, other sequences of steps may be possible. Therefore, theparticular order of the steps set forth in the specification should notbe construed as limitations on the claims. In addition, the claimsdirected to the method and/or process of the present invention shouldnot be limited to the performance of their steps in the order written,and one skilled in the art can readily appreciate that the sequences maybe varied and still remain within the spirit and scope of the presentinvention.

Example 50 Brain Contusion Results in Up-Regulation of SUR1

Contusion Model: Adult Wistar rats were anesthetized (Ketamine andZylazine) and underwent aseptic surgery to create a right parietalcraniectomy that exposed the dura. A contusion injury was obtained usinga weight-drop device, consisting of an impactor (a thin light rod with a5-mm polypropylene ball at the tip, guided within a glass cylinder) thatwas gently placed on the exposed dura and that was activated by weightdrop (10-gm weight dropped from 2.5 cm). Controls underwent sham surgerythat included craniectomy but no weight drop. Brains were harvested 24hours later and cryosectioned to assess for SUR1 expression usingimmunohistochemistry. The antibody used for immunohistochemistry hadpreviously been shown to be highly specific for SUR1 and to label only asingle band (180 kDa) in the range between 116-290 kDa in peri-infarctbrain tissues (see Simard et al., Nature Medicine, 2006) Immunolabelingshowed prominent up-regulation of SUR1 in the region of contusion (seeFIGS. 47 and 48), consistent with contusion-induced up-regulation ofNCCa-ATP channels.

REFERENCES

All patents and publications cited herein are hereby incorporated byreference in their entirety herein. Full citations for the referencescited herein are provided in the following list.

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

What is claimed is:
 1. A method of ameliorating the effect of areduction in blood flow in peri-infarct brain tissue in ischemic diseaseor injury in a subject comprising administering to the subject an amountof a compound effective to inhibit the activity of a NC_(Ca-ATP) channelin a neuronal cell, a neuroglia cell, or a neural endothelial cell. 2.The method of claim 1, where said compound is a compound capable ofeffecting hypoglycemia in said subject, further comprisingadministration of glucose to said subject effective to at leastpartially ameliorate said hypoglycemic effects in said subject.
 3. Amethod of inhibiting neuronal cell swelling in the brain of a subject,said method comprising administering to the subject a formulationcomprising an effective amount of a compound that blocks the NC_(Ca-ATP)channel and a pharmaceutically acceptable carrier.
 4. The method ofclaim 3, wherein said inhibiting neuronal cell swelling is furtherdefined as preventing neuronal cell swelling.
 5. The method of claim 3,where said formulation comprises a compound capable of effectinghypoglycemia in said subject, further comprising administration ofglucose to said subject effective to at least partially ameliorate saidhypoglycemic effects in said subject.
 6. The method of claim 5, whereinsaid inhibiting neuronal cell swelling is further defined as preventingneuronal cell swelling.
 7. The method of claim 1, wherein theamelioration of the effect of a reduction in blood flow comprises areduction in cytotoxic edema, ionic edema and/or vasogenic edema.
 8. Themethod of claim 1, wherein the neuroglia cell is selected from the groupconsisting of astrocyte, ependymal cell, oligodentrocyte and microglia.9. The method of claim 1, wherein the NC_(Ca-ATP) channel inhibitor is atype 1 sulfonylurea receptor antagonist selected from the groupconsisting of glibenclamide, tolbutamide, repaglinide, nateglinide,meglitinide, midaglizole, LY397364, LY389382, glyclazide, glimepiride,estrogen, estradiol, estrone, estriol, genistein, diethystilbestrol,coumestrol, zearalenone, a compound that inhibits K_(ATP) channels. 10.A method of alleviating brain swelling in a subject, comprisingadministering to the subject a formulation comprising an effectiveamount of a compound that blocks the NC_(Ca-ATP) channel and apharmaceutically acceptable carrier.
 11. The method of claim 10, wheresaid formulation is a formulation capable of effecting hypoglycemia insaid subject, further comprising administration of glucose to saidsubject effective to at least partially ameliorate said hypoglycemiceffects in said subject.
 12. The method of claim 1, wherein compoundeffective to inhibit a NC_(Ca-ATP) channel is administered alimentarily,parenterally, topically, mucosally, or by injection into brainparenchema.
 13. A pharmaceutical composition comprising atherapeutically effective amount of a compound that inhibits aNC_(Ca-ATP) channel or a pharmaceutically acceptable salt thereof,wherein said therapeutically effective amount is effective to ameliorateat least one effect of a reduction in blood flow in peri-infarct braintissue in a subject suffering from ischemic disease in the brain or frombrain injury.
 14. The pharmaceutical composition of claim 13, whereinthe compound that inhibits a NC_(Ca-ATP) channel is selected from thegroup consisting of glibenclamide, tolbutamide, repaglinide,nateglinide, meglitinide, midaglizole, LY397364, LY389382, glyclazide,glimepiride, estrogen, estradiol, estrone, estriol, genistein,diethystilbestrol, coumestrol, and zearalenone.
 15. The pharmaceuticalcomposition of claim 13 wherein the composition further comprisesglucose.
 16. The pharmaceutical composition of claim 15, wherein theamount of said compound that inhibits a NC_(Ca-ATP) channel orpharmaceutically acceptable salt thereof is an amount that has ahypoglycemic effect in a subject to which the pharmaceutical compositionis administered.
 17. The pharmaceutical composition of claim 16, whereinthe amount of said glucose is effective to reduce or eliminate alowering of the blood glucose concentration by said compound orpharmaceutically acceptable salt in the subject to which thepharmaceutical composition is administered.
 18. The pharmaceuticalcomposition of claim 13, wherein the pharmaceutical composition isneuroprotective.
 19. The pharmaceutical composition of claim 15, whereinthe pharmaceutical composition is neuroprotective.
 20. A method oftreating acute cerebral ischemia in a subject comprising administeringto a subject an amount of a compound that inhibits a NC_(Ca-ATP) channelor a pharmaceutically acceptable salt thereof.
 21. The method of claim20, wherein the NC_(Ca-ATP) channel is expressed on neuronal cells,neuroglia cells, neural endothelial cells or a combination thereof. 22.The method of claim 20, wherein said NC_(Ca-ATP) channel inhibitor isselected from the group consisting of glibenclamide, tolbutamide,repaglinide, nateglinide, meglitinide, midaglizole, LY397364, LY389382,glyclazide, glimepiride, estrogen, estradiol, estrone, estriol,genistein, diethystilbestrol, coumestrol, and zearalenone.
 23. Themethod of claim 20, wherein the mode of administration of saidNC_(Ca-ATP) channel inhibitor is selected from the group of modes ofadministration consisting of bolus injection, infusion, and bolusinjection in combination with an infusion.
 24. The method of claim 20,wherein said NC_(Ca-ATP) channel inhibitor is glibenclamide.
 25. Themethod of claim 20, further comprising administering glucose to saidsubject.
 26. The method of claim 25, wherein said NC_(Ca-ATP) channelinhibitor is selected from the group consisting of glibenclamide,tolbutamide, repaglinide, nateglinide, meglitinide, midaglizole,LY397364, LY389382, glyclazide, glimepiride, estrogen, estradiol,estrone, estriol, genistein, diethystilbestrol, coumestrol, andzearalenone.
 27. The method of claim 26, wherein the mode ofadministration of said NC_(Ca-ATP) channel inhibitor is selected fromthe group of modes of administration consisting of bolus injection,infusion, and bolus injection in combination with an infusion.
 28. Themethod of claim 20, wherein compound effective to inhibit a NC_(Ca-ATP)channel is administered alimentarily, parenterally, topically,mucosally, or by injection into brain parenchema.
 29. A neuroprotectiveinfusion kit comprising a compound that inhibits a NC_(Ca-ATP) channelin a neuronal cell, a neuroglia cell, a neural endothelial cell or acombination thereof and an intravenous (IV) infusion solution.
 30. Theneuroprotective infusion kit of claim 29, wherein said IV infusionsolution is an IV infusion solution supplemented with glucose.
 31. Theneuroprotective infusion kit of claim 29 further comprising aneuroprotective bolus kit, wherein the neuroprotective bolus kitcomprises a pre-loaded syringe of a compound that inhibits a NC_(Ca-ATP)channel in a neuronal cell, a neuroglia cell, a neural endothelial cellor a combination thereof within an IV solution.
 32. The kit of claim 31,wherein said IV solution is an IV solution supplemented with glucose.33. A method of preventing neural cell swelling in the brain of asubject, said method comprising administering to the subject aformulation comprising an effective amount of a compound that blocks theNC_(Ca-ATP) channel, glucose, and a pharmaceutically acceptable carrier.34. A method of alleviating one or more effects of traumatic braininjury or cerebral ischemia stemming from neural cell swelling in asubject, comprising administering to the subject a formulationcomprising an effective amount of a compound that blocks the NC_(Ca-ATP)channel, glucose, and a pharmaceutically acceptable carrier.
 35. Amethod of alleviating one or more effects of traumatic brain injury orcerebral ischemia in a subject, comprising administering to the subjecta formulation comprising an effective amount of a sulfonylurea compoundand a pharmaceutically acceptable carrier.